Vaccine composition containing synthetic adjuvant

ABSTRACT

Compositions and methods, including vaccines and pharmaceutical compositions for inducing or enhancing an immune response are disclosed based on the discovery of useful immunological adjuvant properties in a synthetic, glucopyranosyl lipid adjuvant (GLA) that is provided in substantially homogeneous form. Chemically defined, synthetic GLA offers a consistent vaccine component from lot to lot without the fluctuations in contaminants or activity that compromise natural-product adjuvants. Also provided are vaccines and pharmaceutical compositions that include GLA and one or more of an antigen, a Toll-like receptor (TLR) agonist, a co-adjuvant and a carrier such as a pharmaceutical carrier.

CROSS-REFERENCE(S) TO RELATED APPLICATION(S)

This application is a continuation of U.S. patent application Ser. No.12/351,710, filed Jan. 9, 2009, now pending; which is a continuation ofU.S. patent application Ser. No. 12/134,127 filed Jun. 5, 2008, nowabandoned; and a continuation-in-part of U.S. application Ser. No.12/154,663, filed May 22, 2008, now abandoned; and acontinuation-in-part of U.S. application Ser. No. 11/862,122 filed Sep.26, 2007, now pending, which claims the benefit under 35 U.S.C. §119(e)of U.S. Provisional Patent Application No. 60/847,404 filed Sep. 26,2006; all of these applications are incorporated herein by reference intheir entireties.

STATEMENT OF GOVERNMENT INTEREST

This invention was made in part with government support under Grant No.AI-25038 awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of pharmaceutical and vaccinecompositions. More specifically, embodiments described herein relate topharmaceutical and vaccine compositions, as well as related prophylacticand therapeutic methods, wherein the compositions comprise aglucopyranosyl lipid adjuvant (GLA).

2. Description of the Related Art

The immune system of higher organisms has been characterized asdistinguishing foreign agents (or “non-self”) agents from familiar or“self” components, such that foreign agents elicit immune responseswhile “self” components are ignored or tolerated. Immune responses havetraditionally been characterized as either humoral responses, in whichantibodies specific for antigens are produced by differentiated Blymphocytes known as plasma cells, or cell mediated responses, in whichvarious types of T lymphocytes act to eliminate antigens by a number ofmechanisms. For example, CD4+ helper T cells that are capable ofrecognizing specific antigens may respond by releasing soluble mediatorssuch as cytokines to recruit additional cells of the immune system toparticipate in an immune response. Also, CD8+ cytotoxic T cells that arealso capable of specific antigen recognition may respond by binding toand destroying or damaging an antigen-bearing cell or particle. It isknown in the immunological arts to provide certain vaccines according toa variety of formulations, usually for the purpose of inducing a desiredimmune response in a host.

Several strategies for eliciting specific immune responses through theadministration of a vaccine to a host include immunization withheat-killed or with live, attenuated infectious pathogens such asviruses, bacteria or certain eukaryotic pathogens; immunization with anon-virulent infective agent capable of directing the expression ofgenetic material encoding the antigen(s) to which an immune response isdesired; and immunization with subunit vaccines that contain isolatedimmunogens (such as proteins) from a particular pathogen in order toinduce immunity against the pathogen. (See, e.g., Liu, 1998 NatureMedicine 4(5 suppl.):515.) For certain antigens there may be one or moretypes of desirable immunity for which none of these approaches has beenparticularly effective, including the development of vaccines that areeffective in protecting a host immunologically against humanimmunodeficiency viruses or other infectious pathogens, cancer,autoimmune disease, or other clinical conditions.

It has long been known that enterobacterial lipopolysaccharide (LPS) isa potent stimulator of the immune system, although its use in adjuvantshas been curtailed by its toxic effects. A non-toxic derivative of LPS,monophosphoryl lipid A (MPL), produced by removal of the corecarbohydrate group and the phosphate from the reducing-end glucosamine,has been described by Ribi et al (1986, Immunology andImmunopharmacology of Bacterial Endotoxins, Plenum Publ. Corp., NY, p407-419).

A further detoxified version of MPL results from the removal of the acylchain from the 3-position of the disaccharide backbone, and is called3-O-deacylated monophosphoryl lipid A (3D-MPL). It can be purified andprepared by the methods taught in GB 2122204B, which reference alsodiscloses the preparation of diphosphoryl lipid A, and 3-O-deacylatedvariants thereof. For example, 3D-MPL has been prepared in the form ofan emulsion having a small particle size less than 0.2 μm in diameter,and its method of manufacture is disclosed in WO 94/21292. Aqueousformulations comprising monophosphoryl lipid A and a surfactant havebeen described in WO9843670A2.

Bacterial lipopolysaccharide-derived adjuvants to be formulated inadjuvant combinations may be purified and processed from bacterialsources, or alternatively they may be synthetic. For example, purifiedmonophosphoryl lipid A is described in Ribi et at 1986 (supra), and3-O-deacylated monophosphoryl or diphosphoryl lipid A derived fromSalmonella sp. is described in GB 2220211 and U.S. Pat. No. 4,912,094.3D-MPL and the β(1-6) glucosamine disaccharides as well as otherpurified and synthetic lipopolysaccharides have been described (WO98/01139; U.S. Pat. No. 6,005,099 and EP 0 729 473 B1, Hilgers et al.,1986 Int. Arch. Allergy Immunol., 79(4):392-6; Hilgers et at., 1987,Immunology, 60(1); 141-6; and EP 0 549 074 B1). Combinations of 3D-MPLand saponin adjuvants derived from the bark of Quillaja Saponaria molinahave been described in EP 0 761 231B. WO 95/17210 discloses an adjuvantemulsion system based on squalene, α-tocopherol, and polyoxyethylenesorbitan monooleate (TWEEN™-80), formulated with the immunostimulantQS21, and optionally including 3D-MPL. Despite the accessibility of suchcombinations, the use of adjuvants derived from natural products isaccompanied by high production costs, inconsistency from lot to lot,difficulties associated with large-scale production, and uncertaintywith respect to the presence of impurities in the compositional make-upof any given preparation.

Clearly there is a need for improved vaccines, and in particular forvaccines that beneficially contain high-purity, chemically definedadjuvant components that exhibit lot-to-lot consistency and that can bemanufactured efficiently on an industrial scale without introducingunwanted or structurally undefined contaminants. The present inventionprovides compositions and methods for such vaccines, and offers otherrelated advantages.

BRIEF SUMMARY OF THE INVENTION

The present invention in its several embodiments is directed tocompositions and methods that advantageously employ the syntheticglucopyranosyl lipid adjuvant (GLA) as an adjuvant and vaccinecomponent. According to one embodiment of the invention describedherein, there is provided a vaccine composition comprising an antigenand a glucopyranosyl lipid adjuvant (GLA).

In other embodiments there is provided a vaccine composition comprising(a) an antigen; a glucopyranosyl lipid adjuvant (GLA); and a toll-likereceptor (TLR) agonist, wherein in certain further embodiments the TLRagonist is selected from lipopolysaccharide, peptidoglycan, polyl:C,CpG, 3M003, flagellin, Leishmania homolog of eukaryotic ribosomalelongation and initiation factor 4a (LeIF) and at least one hepatitis Cantigen. In another embodiment there is provided a vaccine compositioncomprising: an antigen; a glucopyranosyl lipid adjuvant (GLA); and atleast one co-adjuvant that is selected from saponins and saponinmimetics. In another embodiment there is provided a vaccine compositioncomprising an antigen; a glucopyranosyl lipid adjuvant (GLA); and acarrier that comprises at least one of an oil and ISCOMATRIX™. Inanother embodiment there is provided a vaccine composition comprising anantigen; a glucopyranosyl lipid adjuvant (GLA); and one or more of: (i)at least one co-adjuvant, (ii) at least one TLR agonist, (iii) at leastone imidazoquinoline immune response modifier, and (iv) at least onedouble stem loop immune modifier (dSLIM). In certain further embodiments(i) the co-adjuvant, when present, is selected from alum, a plantalkaloid and a detergent, wherein the plant alkaloid is selected fromtomatine and the detergent is selected from saponin, Polysorbate 80,Span 85 and Stearyl tyrosine, (ii) the TLR agonist, when present, isselected from lipopolysaccharide, peptidoglycan, polyl:C, CpG, 3M003,flagellin, Leishmania homolog of eukaryotic ribosomal elongation andinitiation factor 4a (LeIF) and at least one hepatitis C antigen, and(iii) the imidazoquinoline immune response modifier, when present, isselected from resiquimod (R848), imiquimod and gardiquimod. In anotherembodiment there is provided a vaccine composition comprising: anantigen; a glucopyranosyl lipid adjuvant (GLA); and at least one of aco-adjuvant and a pharmaceutically acceptable carrier, wherein: theco-adjuvant is selected from a cytokine, a detergent, and a blockcopolymer or biodegradable polymer, and the pharmaceutically acceptablecarrier comprises a carrier that is selected from calcium phosphate, anoil-in-water emulsion, a water-in-oil emulsion, a liposome, a novosome,a non-ionic surfactant vesicle (e.g., niosome) and a microparticle. In aparticular embodiment, where a liposome or similar carrier is used, theGLA is in the laminar structure of the liposome or is encapsulated. Inanother particular embodiment, where a microparticle is used, themicroparticle is one that is based on or comprises polymer fat lipids.

In certain further embodiments the cytokine is selected from GM-CSF,IL-2, IL-7, IL-12, TNF-α and IFN-gamma, the block copolymer orbiodegradable polymer is selected from Pluronic L121, CRL1005, PLGA,PLA, PLG, and polyl:C, and the detergent is selected from the groupconsisting of saponin, Polysorbate 80, Span 85 and Stearyl tyrosine.

In other embodiments there is provided a vaccine composition comprising:at least one recombinant expression construct which comprises a promoteroperably linked to a nucleic acid sequence encoding an antigen; and aglucopyranosyl lipid adjuvant (GLA). In one embodiment the recombinantexpression construct is present in a viral vector, which in certainfurther embodiments is present in a virus that is selected from anadenovirus, an adeno-associated virus, a herpesvirus, a lentivirus, apoxvirus, and a retrovirus.

According to certain of any of the above described embodiments, the GLAis not 3′-de-O-acylated. According to certain of any of the abovedescribed embodiments, the GLA comprises: (i) a diglucosamine backbonehaving a reducing terminus glucosamine linked to a non-reducing terminusglucosamine through an ether linkage between hexosamine position 1 ofthe non-reducing terminus glucosamine and hexosamine position 6 of thereducing terminus glucosamine; (ii) an O-phosphoryl group attached tohexosamine position 4 of the non-reducing terminus glucosamine; and(iii) up to six fatty acyl chains; wherein one of the fatty acyl chainsis attached to 3-hydroxy of the reducing terminus glucosamine through anester linkage, wherein one of the fatty acyl chains is attached to a2-amino of the non-reducing terminus glucosamine through an amidelinkage and comprises a tetradecanoyl chain linked to an alkanoyl chainof greater than 12 carbon atoms through an ester linkage, and whereinone of the fatty acyl chains is attached to 3-hydroxy of thenon-reducing terminus glucosamine through an ester linkage and comprisesa tetradecanoyl chain linked to an alkanoyl chain of greater than 12carbon atoms through an ester linkage.

According to certain of any of the above described embodiments thatinclude a TLR agonist, the TLR agonist is capable of delivering abiological signal by interacting with at least one TLR that is selectedfrom TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8 and TLR-9. Incertain further embodiments the TLR agonist is selected fromlipopolysaccharide, peptidoglycan, polyl:C, CpG, 3M003, flagellin,Leishmania homolog of eukaryotic ribosomal elongation and initiationfactor 4a (LeIF) and at least one hepatitis C antigen. In a particularembodiment, where a TLR-7 and/or TLR-8 agonist is used, the TLR-7 and/orTLR-8 agonist is entrapped within a vesicle.

According to certain of any of the above described embodiments, the GLAhas the formula:

where: R¹, R³, R⁵ and R⁶ are C₁₁-C₂₀ alkyl; and R² and R⁴ are C₁₂-C₂₀alkyl.

According to certain of any of the above described embodiments, thevaccine composition is capable of eliciting an immune response in ahost. In certain further embodiments the immune response is specific forthe antigen. According to certain of any of the above describedembodiments, the antigen is capable of eliciting in a host an immuneresponse that is selected from a humoral response and a cell-mediatedresponse. According to certain of any of the above describedembodiments, the vaccine composition is capable of eliciting in a hostat least one immune response that is selected from a T_(H)1-type Tlymphocyte response, a T_(H)2-type T lymphocyte response, a cytotoxic Tlymphocyte (CTL) response, an antibody response, a cytokine response, alymphokine response, a chemokine response, and an inflammatory response.According to certain of any of the above described embodiments, thevaccine composition is capable of eliciting in a host at least oneimmune response that is selected from (a) production of one or aplurality of cytokines wherein the cytokine is selected frominterferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), (b)production of one or a plurality of interleukins wherein the interleukinis selected from IL-1, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12,IL-13, IL-16, IL-18 and IL-23, (c) production one or a plurality ofchemokines wherein the chemokine is selected from MIP-1α, MIP-1β,RANTES, CCL4 and CCL5, and (d) a lymphocyte response that is selectedfrom a memory T cell response, a memory B cell response, an effector Tcell response, a cytotoxic T cell response and an effector B cellresponse.

According to certain of any of the above described embodiments, theantigen is derived from at least one infectious pathogen that isselected from a bacterium, a virus, and a fungus.

In certain further embodiments the bacterium is an Actinobacterium, andin certain still further embodiments the Actinobacterium is amycobacterium. In certain other related embodiments the mycobacterium isselected from M. tuberculosis and M. leprae. In certain other relatedembodiments the bacterium is selected from Salmonella, Neisseria,Borrelia, Chlamydia and Bordetella.

In certain other related embodiments the virus is selected from a herpessimplex virus, a human immunodeficiency virus (HIV), a felineimmunodeficiency virus (FIV), cytomegalovirus, Varicella Zoster Virus,hepatitis virus, Epstein Barr Virus (EBV), respiratory syncytial virus,human papilloma virus (HPV) and a cytomegalovirus. According to certainof any of the above described embodiments, the antigen is derived from ahuman immunodeficiency virus, which in certain further embodiments isselected from HIV-1 and HIV-2.

In certain other related embodiments the fungus is selected fromAspergillus, Blastomyces, Coccidioides and Pneumocystis. In certainother related embodiments the fungus is a yeast, which in certainfurther embodiments is a Candida, wherein in certain still furtherembodiments the Candida is selected from C. albicans, C. glabrata, C.krusei, C. lusitaniae, C. tropicalis and C. parapsilosis.

According to certain of any of the above described embodiments, theantigen is derived from a parasite, which in certain further embodimentsis a protozoan, which in certain further embodiments is a Plasmodium,which in certain still further embodiments is selected from P.falciparum, P. vivax, P. malariae and P. ovale. In certain otherembodiments the parasite is selected from Acanthamoeba, Entamoebahistolytica, Angiostrongylus, Schistosoma mansonii, Schistosomahaematobium, Schistosoma japonicum, Schistosoma mekongi,Cryptosporidium, Ancylostoma, Entamoeba histolytica, Entamoeba coli,Entamoeba dispar, Entamoeba hartmanni, Entamoeba polecki, Wuchereriabancrofti, Giardia, Leishmania, Enterobius vermicularis, Ascarislumbricoides, Trichuris trichuria, Necator americanus, Ancylostomaduodenale, Brugia malayi, Onchocerca volvulus, Dracanculus medinensis,Trichinella spiralis, Strongyloides stercoralis, Opisthorchis sinensis,Paragonimus sp, Fasciola hepatica, Fasciola magna, Fasciola gigantica),Taenia saginata and Taenia solium.

According to certain of any of the above described embodiments, theantigen is derived from at least one cancer cell. In certain furtherembodiments the cancer cell originates in a primary solid tumor, and incertain other embodiments the cancer cell originates in a cancer that isa metastatic or secondary solid tumor, and in certain other embodimentsthe cancer cell originates in a cancer that is a circulating tumor or anascites tumor. In certain related embodiments the cancer cell originatesin a cancer that is selected from cervical cancer, ovarian cancer,breast cancer, prostate cancer, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, pseudomyxoma petitonei,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma and Wilms'tumor. In certain other related embodiments the cancer cell originatesin a cancer that is selected from testicular tumor, lung carcinoma,small cell lung carcinoma, bladder carcinoma, epithelial carcinoma,glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, oliodendroglioma,meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma,multiple myeloma, Waldenstrom's macroglobulinemia and heavy chaindisease.

According to certain of any of the above described embodiments, theantigen is derived from, or is immunologically cross-reactive with, atleast one epitope, biomolecule, cell or tissue that is associated withan autoimmune disease. In certain further embodiments the epitope,biomolecule, cell or tissue that is associated with an autoimmunedisease is selected from snRNP when the autoimmune disease is systemiclupus erythematosus, at least one of thyroglobulin, thyrotropin receptorand a thyroid epithelial cell when the autoimmune disease is Graves'disease, a platelet when the autoimmune disease is thrombocytopenicpurpura, at least one of pemphigus antigen, desmoglein-3, desmoplakin,envoplakin and bullous pemphigoid antigen 1 when the autoimmune diseaseis pemphigus, myelin basic protein when the autoimmune disease ismultiple sclerosis, a pancreatic islet beta cell when the autoimmunedisease is type 1 diabetes, and an acetylcholine receptor when theautoimmune disease is myasthenia gravis.

In another embodiment there is provided a pharmaceutical composition forinducing or enhancing an immune response, comprising a glucopyranosyllipid adjuvant (GLA); and a pharmaceutically acceptable carrier orexcipient. In another embodiment there is provided a pharmaceuticalcomposition for inducing or enhancing an immune response comprising anantigen; a glucopyranosyl lipid adjuvant (GLA); and a pharmaceuticallyacceptable carrier or excipient. In another embodiment there is provideda pharmaceutical composition for inducing or enhancing an immuneresponse comprising an antigen; a glucopyranosyl lipid adjuvant (GLA); atoll-like receptor (TLR) agonist; and a pharmaceutically acceptablecarrier or excipient. In a further embodiment the TLR agonist isselected from lipopolysaccharide, peptidoglycan, polyl:C, CpG, 3M003,flagellin, Leishmania homolog of eukaryotic ribosomal elongation andinitiation factor 4a (LeIF) and at least one hepatitis C antigen. Inanother embodiment there is provided a pharmaceutical composition forinducing or enhancing an immune response comprising: an antigen; aglucopyranosyl lipid adjuvant (GLA); at least one co-adjuvant that isselected from saponins and saponin mimetics; and a pharmaceuticallyacceptable carrier or excipient. In another embodiment there is provideda pharmaceutical composition for inducing or enhancing an immuneresponse comprising antigen; a glucopyranosyl lipid adjuvant (GLA); anda pharmaceutically acceptable carrier that comprises at least one of anoil and ISCOMATRIX™. In another embodiment there is provided apharmaceutical composition for inducing or enhancing an immune responsecomprising: (a) an antigen; (b) a glucopyranosyl lipid adjuvant (GLA);(c) one or more of: (i) at least one co-adjuvant, (ii) at least one TLRagonist, (iii) at least one imidazoquinoline immune response modifier,and (iv) at least one double stem loop immune modifier (dSLIM); and (d)a pharmaceutically acceptable carrier or excipient. In certain furtherembodiments (i) the co-adjuvant, when present, is selected from alum, aplant alkaloid and a detergent, wherein the plant alkaloid is tomatineand the detergent is selected from saponin, Polysorbate 80, Span 85 andStearyl tyrosine, (ii) the TLR agonist, when present, is selected fromlipopolysaccharide, peptidoglycan, polyl:C, CpG, 3M003, flagellin,Leishmania homolog of eukaryotic ribosomal elongation and initiationfactor 4a (LeIF) and at least one hepatitis C antigen, and (iii) theimidazoquinoline immune response modifier, when present, is selectedfrom resiquimod (R848), imiquimod and gardiquimod.

In another embodiment there is provided a pharmaceutical composition forinducing or enhancing an immune response, comprising: an antigen; aglucopyranosyl lipid adjuvant (GLA); and at least one co-adjuvant; and apharmaceutically acceptable carrier, wherein: the co-adjuvant isselected from a cytokine, a block copolymer or biodegradable polymer,and a detergent, and the pharmaceutically acceptable carrier comprises acarrier that is selected from calcium phosphate, an oil-in-wateremulsion, a water-in-oil emulsion, a liposome, and a microparticle. Incertain further embodiments the cytokine is selected from GM-CSF, IL-2,IL-7, IL-12, TNF and IFN-gamma, the block copolymer or biodegradablepolymer is selected from Pluronic® L121, CRL1005, PLGA, PLA, PLG, andpolyl:C, and the detergent is selected from the group consisting ofsaponin, Polysorbate 80, Span 85 and Stearyl tyrosine.

In another embodiment there is provided a pharmaceutical compositioncomprising: at least one recombinant expression construct whichcomprises a promoter operably linked to a nucleic acid sequence encodingan antigen; a glucopyranosyl lipid adjuvant (GLA); and apharmaceutically acceptable carrier or excipient. In certain furtherembodiments the recombinant expression construct is present in a viralvector, which in certain further embodiments is present in a virus thatis selected from an adenovirus, an adeno-associated virus, aherpesvirus, a lentivirus, a poxvirus, and a retrovirus.

According to certain further embodiments of the above-describedpharmaceutical compositions, the antigen and the GLA are in contact withone another, and according to certain other further embodiments of theabove-described pharmaceutical compositions, the antigen and the GLA arenot in contact with one another. In certain further embodiments whereinthe antigen and the GLA are not in contact with one another, they arepresent in separate containers. In other embodiments there is provided apharmaceutical composition for inducing or enhancing an immune responsecomprising a first combination comprising an antigen and a firstpharmaceutically acceptable carrier or excipient; and a secondcombination comprising a glucopyranosyl lipid adjuvant (GLA) and asecond pharmaceutically acceptable carrier or excipient, wherein theantigen and the GLA are not in contact with one another. In a furtherembodiment the antigen and the GLA are present in separate containers.In certain related embodiments the first pharmaceutically acceptablecarrier or excipient is different from the second pharmaceuticallyacceptable carrier or excipient. In other related embodiments the firstpharmaceutically acceptable carrier or excipient is not different fromthe second pharmaceutically acceptable carrier or excipient.

In another embodiment there is provided a method of treating orpreventing an infectious disease in a subject having or suspected ofbeing at risk for having the infectious disease, the method comprisingadministering to the subject a vaccine composition that comprises (a) anantigen; and (b) a glucopyranosyl lipid adjuvant (GLA), wherein theantigen is derived from, or is immunologically cross-reactive with, atleast one infectious pathogen that is associated with the infectiousdisease, and thereby treating or preventing the infectious disease. Inanother embodiment there is provided a method of treating or preventingan infectious disease in a subject having or suspected of being at riskfor having the infectious disease, the method comprising administeringto the subject a vaccine composition that comprises (a) an antigen; (b)a glucopyranosyl lipid adjuvant (GLA); and (c) a toll-like receptor(TLR) agonist, wherein the antigen is derived from, or isimmunologically cross-reactive with, at least one infectious pathogenthat is associated with the infectious disease, and thereby treating orpreventing the infectious disease. In a further embodiment the TLRagonist is selected from lipopolysaccharide, peptidoglycan, polyl:C,CpG, 3M003, flagellin, Leishmania homolog of eukaryotic ribosomalelongation and initiation factor 4a (LeIF) and at least one hepatitis Cantigen. In another embodiment there is provided a method of treating orpreventing an infectious disease in a subject having or suspected ofbeing at risk for having the infectious disease, the method comprisingadministering to the subject a vaccine composition that comprises (a) anantigen; (b) a glucopyranosyl lipid adjuvant (GLA); and (c) at least oneco-adjuvant that is selected from the group consisting of saponins andsaponin mimetics, wherein the antigen is derived from, or isimmunologically cross-reactive with, at least one infectious pathogenthat is associated with the infectious disease, and thereby treating orpreventing the infectious disease. In another embodiment there isprovided a method of treating or preventing an infectious disease in asubject having or suspected of being at risk for having the infectiousdisease, the method comprising administering to the subject a vaccinecomposition that comprises (a) an antigen; (b) a glucopyranosyl lipidadjuvant (GLA); and (c) a carrier that comprises at least one of an oiland ISCOMATRIX™, wherein the antigen is derived from, or isimmunologically cross-reactive with, at least one infectious pathogenthat is associated with the infectious disease, and thereby treating orpreventing the infectious disease. In another embodiment there isprovided a method of treating or preventing an infectious disease in asubject having or suspected of being at risk for having the infectiousdisease, the method comprising administering to the subject a vaccinecomposition that comprises (a) an antigen; (b) a glucopyranosyl lipidadjuvant (GLA); and (c) one or more of: (i) at least one co-adjuvant,(ii) at least one TLR agonist, (iii) at least one imidazoquinolineimmune response modifier, and (iv) at least one double stem loop immunemodifier (dSLIM), wherein the antigen is derived from, or isimmunologically cross-reactive with, at least one infectious pathogenthat is associated with the infectious disease, and thereby treating orpreventing the infectious disease. In certain further embodiments, (i)the co-adjuvant, when present, is selected from alum, a plant alkaloidand a detergent, wherein the plant alkaloid is tomatine and thedetergent is selected from saponin, Polysorbate 80, Span 85 and Stearyltyrosine, (ii) the TLR agonist, when present, is selected from the groupconsisting of lipopolysaccharide, peptidoglycan, polyl:C, CpG, 3M003,flagellin, Leishmania homolog of eukaryotic ribosomal elongation andinitiation factor 4a (LeIF) and at least one hepatitis C antigen, and(iii) the imidazoquinoline immune response modifier, when present, isselected from the group consisting of resiquimod (R848), imiquimod andgardiquimod.

In another embodiment there is provided a method of treating orpreventing an infectious disease in a subject having or suspected ofbeing at risk for having the infectious disease, the method comprisingadministering to the subject a vaccine composition that comprises (a) anantigen; (b) a glucopyranosyl lipid adjuvant (GLA); and (c) at least oneof a co-adjuvant and a pharmaceutically acceptable carrier, wherein: theco-adjuvant is selected from a cytokine, a block copolymer orbiodegradable polymer, and a detergent, and the pharmaceuticallyacceptable carrier comprises a carrier that is selected from the groupconsisting of calcium phosphate, an oil-in-water emulsion, awater-in-oil emulsion, a liposome, and a microparticle, wherein theantigen is derived from, or is immunologically cross-reactive with, atleast one infectious pathogen that is associated with the infectiousdisease, and thereby treating or preventing the infectious disease. Incertain further embodiments the cytokine is selected from GM-CSF, IL-2,IL-7, IL-12, TNF-α and IFN-gamma, the block copolymer or biodegradablepolymer is selected from Pluronic L121, CRL1005, PLGA, PLA, PLG, andpolyl:C, and the detergent is selected from the group consisting ofsaponin, Polysorbate 80, Span 85 and Stearyl tyrosine.

In another embodiment there is provided a method of treating orpreventing an infectious disease in a subject having or suspected ofbeing at risk for having the infectious disease, the method comprisingadministering to the subject a vaccine composition that comprises (a) atleast one recombinant expression construct which comprises a promoteroperably linked to a nucleic acid sequence encoding an antigen; and (b)a glucopyranosyl lipid adjuvant (GLA), wherein the antigen is derivedfrom, or is immunologically cross-reactive with, at least one infectiouspathogen that is associated with the infectious disease, and therebytreating or preventing the infectious disease. In a further embodimentthe recombinant expression construct is present in a viral vector, whichin certain still further embodiments is present in a virus that isselected from an adenovirus, an adeno-associated virus, a herpesvirus, alentivirus, a poxvirus, and a retrovirus. According to certainembodiments relating to the above described methods, the antigen isderived from at least one infectious pathogen that is selected from abacterium, a virus, and a fungus.

In another embodiment there is provided a method of treating orpreventing autoimmune disease in a subject having or suspected of beingat risk for having an autoimmune disease, the method comprisingadministering to the subject a vaccine composition that comprises (a) anantigen; and (b) a glucopyranosyl lipid adjuvant (GLA), wherein theantigen is derived from, or is immunologically cross-reactive with, atleast one epitope, biomolecule, cell or tissue that is associated withthe autoimmune disease, and thereby treating or preventing theautoimmune disease. In another embodiment there is provided a method oftreating or preventing an autoimmune disease in a subject having orsuspected of being at risk for having an autoimmune disease, the methodcomprising administering to the subject a vaccine composition thatcomprises (a) an antigen; (b) a glucopyranosyl lipid adjuvant (GLA); and(c) a toll-like receptor (TLR) agonist, wherein the antigen is derivedfrom, or is immunologically cross-reactive with, at least one epitope,biomolecule, cell or tissue that is associated with the autoimmunedisease, and thereby treating or preventing the autoimmune disease. Incertain further embodiments the TLR agonist is selected fromlipopolysaccharide, peptidoglycan, polyl:C, CpG, 3M003, flagellin,Leishmania homolog of eukaryotic ribosomal elongation and initiationfactor 4a (LeIF) and at least one hepatitis C antigen. In anotherembodiment there is provided a method of treating or preventing anautoimmune disease in a subject having or suspected of being at risk forhaving an autoimmune disease, the method comprising administering to thesubject a vaccine composition that comprises (a) an antigen; (b) aglucopyranosyl lipid adjuvant (GLA); and (c) at least one co-adjuvantthat is selected from the group consisting of saponins and saponinmimetics, wherein the antigen is derived from, or is immunologicallycross-reactive with, at least one epitope, biomolecule, cell or tissuethat is associated with the autoimmune disease, and thereby treating orpreventing the autoimmune disease. In another embodiment there isprovided a method of treating or preventing an autoimmune disease in asubject having or suspected of being at risk for having an autoimmunedisease, the method comprising administering to the subject a vaccinecomposition that comprises (a) an antigen; (b) a glucopyranosyl lipidadjuvant (GLA); and (c) a carrier that comprises at least one of an oiland ISCOMATRIX™, wherein the antigen is derived from, or isimmunologically cross-reactive with, at least one epitope, biomolecule,cell or tissue that is associated with the autoimmune disease, andthereby treating or preventing the autoimmune disease. In anotherembodiment there is provided a method of treating or preventing anautoimmune disease in a subject having or suspected of being at risk forhaving an autoimmune disease, the method comprising administering to thesubject a vaccine composition that comprises (a) an antigen; (b) aglucopyranosyl lipid adjuvant (GLA); and (c) one or more of: (i) atleast one co-adjuvant, (ii) at least one TLR agonist, (iii) at least oneimidazoquinoline immune response modifier, and (iv) at least one doublestem loop immune modifier (dSLIM), wherein the antigen is derived from,or is immunologically cross-reactive with, at least one epitope,biomolecule, cell or tissue that is associated with the autoimmunedisease, and thereby treating or preventing the autoimmune disease. Incertain further embodiments (i) the co-adjuvant, when present, isselected from alum, a plant alkaloid and a detergent, wherein the plantalkaloid is tomatine and the detergent is selected from saponin,Polysorbate 80, Span 85 and Stearyl tyrosine, (ii) the TLR agonist, whenpresent, is selected from the group consisting of lipopolysaccharide,peptidoglycan, polyl:C, CpG, 3M003, flagellin, Leishmania homolog ofeukaryotic ribosomal elongation and initiation factor 4a (LeIF) and atleast one hepatitis C antigen, and (iii) the imidazoquinoline immuneresponse modifier, when present, is selected from the group consistingof resiquimod (R848), imiquimod and gardiquimod.

In another embodiment there is provided a method of treating orpreventing an autoimmune disease in a subject having or suspected ofbeing at risk for having an autoimmune disease, the method comprisingadministering to the subject a vaccine composition that comprises (a) anantigen; (b) a glucopyranosyl lipid adjuvant (GLA); and (c) at least oneof a co-adjuvant and a pharmaceutically acceptable carrier, wherein: theco-adjuvant is selected from a cytokine, a block copolymer orbiodegradable polymer, and a detergent, and the pharmaceuticallyacceptable carrier comprises a carrier that is selected from the groupconsisting of calcium phosphate, an oil-in-water emulsion, awater-in-oil emulsion, a liposome, and a microparticle, wherein theantigen is derived from, or is immunologically cross-reactive with, atleast one epitope, biomolecule, cell or tissue that is associated withthe autoimmune disease, and thereby treating or preventing theautoimmune disease. In a further embodiment the cytokine is selectedfrom GM-CSF, IL-2, IL-7, IL-12, TNF-α and IFN-gamma, the block copolymeror biodegradable polymer is selected from Pluronic L121, CRL1005, PLGA,PLA, PLG, and polyl:C, and the detergent is selected from the groupconsisting of saponin, Polysorbate 80, Span 85 and Stearyl tyrosine.

In another embodiment there is provided a method of treating orpreventing an autoimmune disease in a subject having or suspected ofbeing at risk for having an autoimmune disease, the method comprisingadministering to the subject a vaccine composition that comprises (a) atleast one recombinant expression construct which comprises a promoteroperably linked to a nucleic acid sequence encoding an antigen; and (b)a glucopyranosyl lipid adjuvant (GLA), wherein the antigen is derivedfrom, or is immunologically cross-reactive with, at least one epitope,biomolecule, cell or tissue that is associated with the autoimmunedisease, and thereby treating or preventing the autoimmune disease. In afurther embodiment the recombinant expression construct is present in aviral vector, which in certain further embodiments is present in a virusthat is selected from an adenovirus, an adeno-associated virus, aherpesvirus, a lentivirus, a poxvirus, and a retrovirus.

In certain of the above described embodiments as relate to a method oftreating or preventing an autoimmune disease, the autoimmune disease isselected from Type 1 diabetes, rheumatoid arthritis, multiple sclerosis,systemic lupus erythematosus, myasthenia gravis, Crohn's disease,Graves' disease, thrombocytopenic purpura and pemphigus. In certainother of the above described embodiments as relate to a method oftreating or preventing an autoimmune disease, the epitope, biomolecule,cell or tissue that is associated with an autoimmune disease is selectedfrom snRNP when the autoimmune disease is systemic lupus erythematosus,at least one of thyroglobulin, thyrotropin receptor and a thyroidepithelial cell when the autoimmune disease is Graves' disease, aplatelet when the autoimmune disease is thrombocytopenic purpura, atleast one of pemphigus antigen, desmoglein-3, desmoplakin, envoplakinand bullous pemphigoid antigen 1 when the autoimmune disease ispemphigus, myelin basic protein when the autoimmune disease is multiplesclerosis, a pancreatic islet beta cell when the autoimmune disease istype 1 diabetes, and an acetylcholine receptor when the autoimmunedisease is myasthenia gravis.

According to other embodiments there is provided a method of treating orpreventing cancer in a subject having or suspected of being at risk forhaving an cancer, the method comprising administering to the subject avaccine composition that comprises (a) an antigen; and (b) aglucopyranosyl lipid adjuvant (GLA), wherein the antigen is derivedfrom, or is immunologically cross-reactive with, at least one epitope,biomolecule, cell or tissue that is associated with the cancer, andthereby treating or preventing the cancer. According to otherembodiments there is provided a method of treating or preventing cancerin a subject having or suspected of being at risk for having cancer, themethod comprising administering to the subject a vaccine compositionthat comprises (a) an antigen; (b) a glucopyranosyl lipid adjuvant(GLA); and (c) a toll-like receptor (TLR) agonist, wherein the antigenis derived from, or is immunologically cross-reactive with, at least oneepitope, biomolecule, cell or tissue that is associated with the cancer,and thereby treating or preventing the cancer. In certain furtherembodiments the TLR agonist is selected from lipopolysaccharide,peptidoglycan, polyl:C, CpG, 3M003, flagellin, Leishmania homolog ofeukaryotic ribosomal elongation and initiation factor 4a (LeIF) and atleast one hepatitis C antigen. According to other embodiments there isprovided a method of treating or preventing cancer in a subject havingor suspected of being at risk for having cancer, the method comprisingadministering to the subject a vaccine composition that comprises (a) anantigen; (b) a glucopyranosyl lipid adjuvant (GLA); and (c) at least oneco-adjuvant that is selected from the group consisting of saponins andsaponin mimetics, wherein the antigen is derived from, or isimmunologically cross-reactive with, at least one epitope, biomolecule,cell or tissue that is associated with the cancer, and thereby treatingor preventing the cancer.

According to other embodiments there is provided a method of treating orpreventing cancer in a subject having or suspected of being at risk forhaving cancer, the method comprising administering to the subject avaccine composition that comprises (a) an antigen; (b) a glucopyranosyllipid adjuvant (GLA); and (c) a carrier that comprises at least one ofan oil and ISCOMATRIX™, wherein the antigen is derived from, or isimmunologically cross-reactive with, at least one epitope, biomolecule,cell or tissue that is associated with the cancer, and thereby treatingor preventing the cancer. According to other embodiments there isprovided a method of treating or preventing cancer in a subject havingor suspected of being at risk for having cancer, the method comprisingadministering to the subject a vaccine composition that comprises (a) anantigen; (b) a glucopyranosyl lipid adjuvant (GLA); and (c) one or moreof: (i) at least one co-adjuvant, (ii) at least one TLR agonist, (iii)at least one imidazoquinoline immune response modifier, and (iv) atleast one double stem loop immune modifier (dSLIM), wherein the antigenis derived from, or is immunologically cross-reactive with, at least oneepitope, biomolecule, cell or tissue that is associated with the cancer,and thereby treating or preventing the cancer. In certain furtherembodiments (i) the co-adjuvant, when present, is selected from thegroup consisting of alum, a plant alkaloid and a detergent, wherein theplant alkaloid is tomatine and the detergent is selected from saponin,Polysorbate 80, Span 85 and Stearyl tyrosine, (ii) the TLR agonist, whenpresent, is selected from the group consisting of lipopolysaccharide,peptidoglycan, polyl:C, CpG, 3M003, flagellin, Leishmania homolog ofeukaryotic ribosomal elongation and initiation factor 4a (LeIF) and atleast one hepatitis C antigen, and (iii) the imidazoquinoline immuneresponse modifier, when present, is selected from the group consistingof resiquimod (R848), imiquimod and gardiquimod. According to otherembodiments there is provided a method of treating or preventing cancerin a subject having or suspected of being at risk for having cancer, themethod comprising administering to the subject a vaccine compositionthat comprises (a) an antigen; (b) a glucopyranosyl lipid adjuvant(GLA); and (c) at least one of a co-adjuvant and a pharmaceuticallyacceptable carrier, wherein the co-adjuvant is selected from the groupconsisting of a cytokine, a block copolymer or biodegradable polymer,and a detergent, and the pharmaceutically acceptable carrier comprises acarrier that is selected from the group consisting of calcium phosphate,an oil-in-water emulsion, a water-in-oil emulsion, a liposome, and amicroparticle, wherein the antigen is derived from, or isimmunologically cross-reactive with, at least one epitope, biomolecule,cell or tissue that is associated with the cancer, and thereby treatingor preventing the cancer. In a further embodiment the cytokine isselected from GM-CSF, IL-2, IL-7, IL-12, TNF-α and IFN-gamma, the blockcopolymer or biodegradable polymer is selected from Pluronic L121,CRL1005, PLGA, PLA, PLG, and polyl:C, and the detergent is selected fromsaponin, Polysorbate 80, Span 85 and Stearyl tyrosine. According toother embodiments there is provided a method of treating or preventingcancer in a subject having or suspected of being at risk for havingcancer, the method comprising administering to the subject a vaccinecomposition that comprises (a) at least one recombinant expressionconstruct which comprises a promoter operably linked to a nucleic acidsequence encoding an antigen; and (b) a glucopyranosyl lipid adjuvant(GLA), wherein the antigen is derived from, or is immunologicallycross-reactive with, at least one epitope, biomolecule, cell or tissuethat is associated with the cancer, and thereby treating or preventingthe cancer. In a further embodiment the recombinant expression constructis present in a viral vector, which in certain further embodiments ispresent in a virus that is selected from an adenovirus, anadeno-associated virus, a herpesvirus, a lentivirus, a poxvirus, and aretrovirus.

In certain further embodiments of the above described methods oftreating or preventing cancer the antigen is derived from at least onecancer cell, which in certain further embodiments originates in aprimary solid tumor, and in certain other further embodiments originatesin a cancer that is a metastatic or secondary solid tumor, and incertain other further embodiments originates in a cancer that is acirculating tumor or an ascites tumor. In certain embodiments the cancercell originates in a cancer that is selected from cervical cancer,ovarian cancer, breast cancer, prostate cancer, fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma, pseudomyxomapetitonei, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing'stumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreaticcancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma and Wilms'tumor. In certain other embodiments the cancer cell originates in acancer that is selected from testicular tumor, lung carcinoma, smallcell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma,astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oliodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, multiplemyeloma, Waldenstrom's macroglobulinemia and heavy chain disease.

According to certain further embodiments of any one of theabove-described methods of treating or preventing infectious disease orautoimmune disease or cancer, the step of administering is performedonce, while in certain other further embodiments of such methods thestep of administering is performed at least two times, and in certainother further embodiments the step of administering is performed atleast three times, and in certain other further embodiments the step ofadministering is performed four or more times. According to certainfurther embodiments of any one of the above-described methods oftreating or preventing infectious disease or autoimmune disease orcancer, prior to the step of administering, the subject is primed with apriming agent that is selected from a bacterial extract, a live virusvaccine, at least one recombinant expression construct which comprises apromoter operably linked to a nucleic acid sequence encoding theantigen, and a viral vector that comprises a promoter operably linked toa nucleic acid sequence encoding the antigen. In a further embodimentthe bacterial extract is derived from Bacillus Calmet-Guerin (BCG).

In another embodiment there is provided a method of eliciting orenhancing a desired antigen-specific immune response in a subject,comprising administering to the subject a vaccine composition thatcomprises (a) an antigen, and (b) a glucopyranosyl lipid adjuvant (GLA).In another embodiment there is provided a method of eliciting orenhancing a desired antigen-specific immune response in a subject,comprising administering to the subject a vaccine composition thatcomprises (a) an antigen, (b) a glucopyranosyl lipid adjuvant (GLA), and(c) a toll-like receptor (TLR) agonist. In certain further embodimentsthe TLR agonist is selected from lipopolysaccharide, peptidoglycan,polyl:C, CpG, 3M003, flagellin, Leishmania homolog of eukaryoticribosomal elongation and initiation factor 4a (LeIF) and at least onehepatitis C antigen. In another embodiment there is provided a method ofeliciting or enhancing a desired antigen-specific immune response in asubject, comprising administering to the subject a vaccine compositionthat comprises (a) an antigen, (b) a glucopyranosyl lipid adjuvant(GLA), and (c) at least one co-adjuvant that is selected from the groupconsisting of saponins and saponin mimetics. In another embodiment thereis provided a method of eliciting or enhancing a desiredantigen-specific immune response in a subject, comprising administeringto the subject a vaccine composition that comprises (a) an antigen, (b)a glucopyranosyl lipid adjuvant (GLA), and (c) a carrier that comprisesat least one of an oil and ISCOMATRIX™. In another embodiment there isprovided a method of eliciting or enhancing a desired antigen-specificimmune response in a subject, comprising administering to the subject avaccine composition that comprises (a) an antigen; (b) a glucopyranosyllipid adjuvant (GLA); and (c) one or more of: (i) at least oneco-adjuvant, (ii) at least one TLR agonist, (iii) at least oneimidazoquinoline immune response modifier, and (iv) at least one doublestem loop immune modifier (dSLIM). In certain further embodiments, theco-adjuvant, when present, is selected from alum, a plant alkaloid and adetergent, wherein the plant alkaloid is selected from tomatine and thedetergent is selected from saponin, Polysorbate 80, Span 85 and Stearyltyrosine, (ii) the TLR agonist, when present, is selected from the groupconsisting of lipopolysaccharide, peptidoglycan, polyl:C, CpG, 3M003,flagellin, Leishmania homolog of eukaryotic ribosomal elongation andinitiation factor 4a (LeIF) and at least one hepatitis C antigen, and(iii) the imidazoquinoline immune response modifier, when present, isselected from the group consisting of resiquimod (R848), imiquimod andgardiquimod.

In another embodiment there is provided a method of eliciting orenhancing a desired antigen-specific immune response in a subject,comprising administering to the subject a vaccine composition thatcomprises (a) an antigen; (b) a glucopyranosyl lipid adjuvant (GLA); and(c) at least one of a co-adjuvant and a pharmaceutically acceptablecarrier, wherein: the co-adjuvant is selected from a cytokine, a blockcopolymer, a biodegradable polymer, and a detergent, and thepharmaceutically acceptable carrier comprises a carrier that is selectedfrom calcium phosphate, an oil-in-water emulsion, a water-in-oilemulsion, a liposome, and a microparticle. In certain furtherembodiments the cytokine is selected from GM-CSF, IL-2, IL-7, IL-12,TNF-α and IFN-gamma, the block copolymer or biodegradable polymer isselected from Pluronic L121, CRL1005, PLGA, PLA, PLG, and polyl:C, andthe detergent is selected from the group consisting of saponin,Polysorbate 80, Span 85 and Stearyl tyrosine.

In another embodiment there is provided a method of eliciting orenhancing a desired antigen-specific immune response in a subject,comprising administering to the subject a vaccine composition thatcomprises (a) at least one recombinant expression construct whichcomprises a promoter operably linked to a nucleic acid sequence encodingan antigen, and (b) a glucopyranosyl lipid adjuvant (GLA). In certainfurther embodiments the recombinant expression construct is present in aviral vector, which in certain further embodiments is present in a virusthat is selected from an adenovirus, an adeno-associated virus, aherpesvirus, a lentivirus, a poxvirus, and a retrovirus.

In certain further embodiments of the above described methods ofeliciting or enhancing a desired antigen-specific response in a subject,the GLA is not 3′-de-O-acylated. In certain other further embodiments ofthe above described methods of eliciting or enhancing a desiredantigen-specific response in a subject, the GLA comprises:(i) adiglucosamine backbone having a reducing terminus glucosamine linked toa non-reducing terminus glucosamine through an ether linkage betweenhexosamine position 1 of the non-reducing terminus glucosamine andhexosamine position 6 of the reducing terminus glucosamine; (ii) anO-phosphoryl group attached to hexosamine position 4 of the non-reducingterminus glucosamine; and (iii) up to six fatty acyl chains; wherein oneof the fatty acyl chains is attached to 3-hydroxy of the reducingterminus glucosamine through an ester linkage, wherein one of the fattyacyl chains is attached to a 2-amino of the non-reducing terminusglucosamine through an amide linkage and comprises a tetradecanoyl chainlinked to an alkanoyl chain of greater than 12 carbon atoms through anester linkage, and wherein one of the fatty acyl chains is attached to3-hydroxy of the non-reducing terminus glucosamine through an esterlinkage and comprises a tetradecanoyl chain linked to an alkanoyl chainof greater than 12 carbon atoms through an ester linkage. In certainrelated further embodiments the TLR agonist, when present, is capable ofdelivering a biological signal by interacting with at least one TLR thatis selected from TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8 andTLR-9. In certain further embodiments the TLR agonist is selected fromlipopolysaccharide, peptidoglycan, polyl:C, CpG, 3M003, flagellin,Leishmania homolog of eukaryotic ribosomal elongation and initiationfactor 4a (LeIF) and at least one hepatitis C antigen.

In certain further embodiments of the above described methods ofeliciting or enhancing a desired antigen-specific response in a subject,the GLA has the formula:

where:

-   -   R¹, R³, R⁵ and R⁶ are C₁₁-C₂₀ alkyl; and    -   R² and R⁴ are C₁₂-C₂₀ alkyl.

In certain further embodiments of the above described methods ofeliciting or enhancing a desired antigen-specific response in a subject,the vaccine composition is capable of eliciting an immune response in ahost. In certain further embodiments the immune response is specific forthe antigen. In certain further embodiments of the above describedmethods of eliciting or enhancing a desired antigen-specific response ina subject, the antigen is capable of eliciting in a host an immuneresponse that is selected from a humoral response and a cell-mediatedresponse. In certain further embodiments of the above described methodsof eliciting or enhancing a desired antigen-specific response in asubject, the vaccine composition is capable of eliciting in a host atleast one immune response that is selected from the group consisting of:a T_(H)1-type T lymphocyte response, a T_(H)2-type T lymphocyteresponse, a cytotoxic T lymphocyte (CTL) response, an antibody response,a cytokine response, a lymphokine response, a chemokine response, and aninflammatory response. In certain further embodiments of the abovedescribed methods of eliciting or enhancing a desired antigen-specificresponse in a subject, the vaccine composition is capable of elicitingin a host at least one immune response that is selected from the groupconsisting of: (a) production of one or a plurality of cytokines whereinthe cytokine is selected from the group consisting of interferon-gamma(IFN-γ) and tumor necrosis factor-alpha (TNF-α), (b) production of oneor a plurality of interleukins wherein the interleukin is selected fromIL-1, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-16, IL-18and IL-23, (c) production one or a plurality of chemokines wherein thechemokine is selected from MIP-1α, MIP-1β, RANTES, CCL4 and CCL5, and(d) a lymphocyte response that is selected from a memory T cellresponse, a memory B cell response, an effector T cell response, acytotoxic T cell response and an effector B cell response.

According to certain other embodiments, there is provided a method ofpreparing a vaccine composition, comprising admixing (a) an antigen and(b) a glucopyranosyl lipid adjuvant (GLA). According to certain otherembodiments, there is provided a method of preparing a vaccinecomposition, comprising admixing (a) an antigen, (b) a glucopyranosyllipid adjuvant (GLA) and (c) a toll-like receptor (TLR) agonist. Incertain further embodiments the TLR agonist is selected fromlipopolysaccharide, peptidoglycan, polyl:C, CpG, 3M003, flagellin,Leishmania homolog of eukaryotic ribosomal elongation and initiationfactor 4a (LeIF) and at least one hepatitis C antigen. According tocertain other embodiments, there is provided a method of preparing avaccine composition, comprising admixing (a) an antigen, (b) aglucopyranosyl lipid adjuvant (GLA), and (c) at least one co-adjuvantthat is selected from the group consisting of saponins and saponinmimetics. According to certain other embodiments, there is provided amethod of preparing a vaccine composition, comprising admixing (a) anantigen, (b) a glucopyranosyl lipid adjuvant (GLA), and (c) a carrierthat comprises at least one of an oil and ISCOMATRIX™. According tocertain other embodiments, there is provided a method of preparing avaccine composition, comprising admixing (a) an antigen; (b) aglucopyranosyl lipid adjuvant (GLA); and (c) one or more of: (i) atleast one co-adjuvant, (ii) at least one TLR agonist, (iii) at least oneimidazoquinoline immune response modifier, and (iv) at least one doublestem loop immune modifier (dSLIM). In certain further embodiments, (i)the co-adjuvant, when present, is selected from the group consisting ofalum, a plant alkaloid and a detergent, wherein the plant alkaloid isselected from tomatine and the detergent is selected from saponin,Polysorbate 80, Span 85 and Stearyl tyrosine, (ii) the TLR agonist, whenpresent, is selected from the group consisting of lipopolysaccharide,peptidoglycan, polyl:C, CpG, 3M003, flagellin, Leishmania homolog ofeukaryotic ribosomal elongation and initiation factor 4a (LeIF) and atleast one hepatitis C antigen, and (iii) the imidazoquinoline immuneresponse modifier, when present, is selected from the group consistingof resiquimod (R848), imiquimod and gardiquimod. According to certainother embodiments, there is provided a method of preparing a vaccinecomposition, comprising admixing (a) an antigen; (b) a glucopyranosyllipid adjuvant (GLA); and (c) at least one of a co-adjuvant and apharmaceutically acceptable carrier, wherein: the co-adjuvant isselected from the group consisting of a cytokine, a block copolymer orbiodegradable polymer, and a detergent, and the pharmaceuticallyacceptable carrier comprises a carrier that is selected from the groupconsisting of calcium phosphate, an oil-in-water emulsion, awater-in-oil emulsion, a liposome, and a microparticle. In certainfurther embodiments the cytokine is selected from GM-CSF, IL-2, IL-7,IL-12, TNF-α and IFN-gamma, the block copolymer or biodegradable polymeris selected from Pluronic L121, CRL1005, PLGA, PLA, PLG, and polyl:C,and the detergent is selected from saponin, Polysorbate 80, Span 85 andStearyl tyrosine.

According to certain other embodiments, there is provided a method ofpreparing a vaccine composition, comprising admixing (a) at least onerecombinant expression construct which comprises a promoter operablylinked to a nucleic acid sequence encoding an antigen, and (b) aglucopyranosyl lipid adjuvant (GLA). In certain further embodiments therecombinant expression construct is present in a viral vector, which incertain further embodiments is present in a virus that is selected froman adenovirus, an adeno-associated virus, a herpesvirus, a lentivirus, apoxvirus, and a retrovirus. In certain embodiments the GLA is not3′-de-O-acylated. In certain embodiments the GLA comprises: (i) adiglucosamine backbone having a reducing terminus glucosamine linked toa non-reducing terminus glucosamine through an ether linkage betweenhexosamine position 1 of the non-reducing terminus glucosamine andhexosamine position 6 of the reducing terminus glucosamine; (ii) anO-phosphoryl group attached to hexosamine position 4 of the non-reducingterminus glucosamine; and (iii) up to six fatty acyl chains; wherein oneof the fatty acyl chains is attached to 3-hydroxy of the reducingterminus glucosamine through an ester linkage, wherein one of the fattyacyl chains is attached to a 2-amino of the non-reducing terminusglucosamine through an amide linkage and comprises a tetradecanoyl chainlinked to an alkanoyl chain of greater than 12 carbon atoms through anester linkage, and wherein one of the fatty acyl chains is attached to3-hydroxy of the non-reducing terminus glucosamine through an esterlinkage and comprises a tetradecanoyl chain linked to an alkanoyl chainof greater than 12 carbon atoms through an ester linkage. In certainembodiments the TLR agonist is capable of delivering a biological signalby interacting with at least one TLR that is selected from TLR-2, TLR-3,TLR-4, TLR-5, TLR-6, TLR-7, TLR-8 and TLR-9. In certain furtherembodiments the TLR agonist is selected from lipopolysaccharide,peptidoglycan, polyl:C, CpG, 3M003, flagellin, Leishmania homolog ofeukaryotic ribosomal elongation and initiation factor 4a (LeIF) and atleast one hepatitis C antigen.

According to certain embodiments of the above-described methods ofpreparing a vaccine composition, the GLA has the formula:

where:

-   -   R¹, R³, R⁵ and R⁶ are C₁₁-C₂₀ alkyl; and    -   R² and R⁴ are C₁₂-C₂₀ alkyl.

In certain further embodiments the step of admixing comprisesemulsifying, and in certain other further embodiments the step ofadmixing comprises forming particles, which in certain furtherembodiments comprise microparticles. In certain other furtherembodiments the step of admixing comprises forming a precipitate whichcomprises all or a portion of the antigen and all or a portion of theGLA.

In certain other embodiments there is provided an immunological adjuvantpharmaceutical composition comprising: a glycopyranosyl lipid adjuvant(GLA); and a pharmaceutically acceptable carrier or excipient. Incertain other embodiments there is provided an immunological adjuvantcomposition comprising a glycopyranosyl lipid adjuvant (GLA); and atoll-like receptor (TLR) agonist. In certain further embodiments the TLRagonist is selected from lipopolysaccharide, peptidoglycan, polyl:C,CpG, 3M003, flagellin, Leishmania homolog of eukaryotic ribosomalelongation and initiation factor 4a (LeIF) and at least one hepatitis Cantigen. In certain other embodiments there is provided an immunologicaladjuvant composition comprising: a glycopyranosyl lipid adjuvant (GLA);and at least one co-adjuvant that is selected from saponins and saponinmimetics. In certain other embodiments there is provided animmunological adjuvant pharmaceutical composition comprising: aglycopyranosyl lipid adjuvant (GLA); and a pharmaceutically acceptablecarrier that comprises at least one of an oil and ISCOMATRIX™. Incertain other embodiments there is provided an immunological adjuvantcomposition comprising: (a) a glycopyranosyl lipid adjuvant (GLA); and(b) one or more of: (i) at least one co-adjuvant, (ii) at least one TLRagonist, (iii) at least one imidazoquinoline immune response modifier,and (iv) at least one double stem loop immune modifier (dSLIM).

In certain further embodiments, (i) the co-adjuvant, when present, isselected from the group consisting of alum, a plant alkaloid and adetergent, wherein the plant alkaloid is tomatine and the detergent isselected from saponin, Polysorbate 80, Span 85 and Stearyl tyrosine,(ii) the TLR agonist, when present, is selected from the groupconsisting of lipopolysaccharide, peptidoglycan, polyl:C, CpG, 3M003,flagellin, Leishmania homolog of eukaryotic ribosomal elongation andinitiation factor 4a (LeIF) and at least one hepatitis C antigen, and(iii) the imidazoquinoline immune response modifier, when present, isselected from the group consisting of resiquimod (R848), imiquimod andgardiquimod.

In certain other embodiments there is provided an immunological adjuvantcomposition comprising: a glycopyranosyl lipid adjuvant (GLA); and atleast one of a co-adjuvant and a pharmaceutically acceptable carrier,wherein: the co-adjuvant is selected from the group consisting of acytokine, a block copolymer or biodegradable polymer, and a detergent,and the pharmaceutically acceptable carrier comprises a carrier that isselected from calcium phosphate, an oil-in-water emulsion, awater-in-oil emulsion, a liposome, and a microparticle. In certainfurther embodiments the cytokine is selected from GM-CSF, IL-2, IL-7,IL-12, TNF and IFN-gamma, the block copolymer or biodegradable polymeris selected from Pluronic L121, CRL1005, PLGA, PLA, PLG, and polyl:C,and the detergent is selected from the group consisting of saponin,Polysorbate 80, Span 85 and Stearyl tyrosine.

In certain other embodiments there is provided a method of alteringimmunological responsiveness in a host, comprising: administering to thehost an immunological adjuvant pharmaceutical composition that comprisesa glycopyranosyl lipid adjuvant (GLA), and a pharmaceutically acceptablecarrier or excipient, and thereby altering host immunologicalresponsiveness. In certain other embodiments there is provided a methodof altering immunological responsiveness in a host, comprising:administering to the host an immunological adjuvant composition thatcomprises a glycopyranosyl lipid adjuvant (GLA), and (b) a toll-likereceptor (TLR) agonist, and thereby altering host immunologicalresponsiveness. In certain further embodiments the TLR agonist isselected from lipopolysaccharide, peptidoglycan, polyl:C, CpG, 3M003,flagellin, Leishmania homolog of eukaryotic ribosomal elongation andinitiation factor 4a (LeIF) and at least one hepatitis C antigen. Incertain other embodiments there is provided a method of alteringimmunological responsiveness in a host, comprising: administering to thehost an immunological adjuvant composition that comprises aglycopyranosyl lipid adjuvant (GLA), and at least one co-adjuvant thatis selected from the group consisting of saponins and saponin mimetics,and thereby altering host immunological responsiveness. In certain otherembodiments there is provided a method of altering immunologicalresponsiveness in a host, comprising: administering to the host animmunological adjuvant composition that comprises a glycopyranosyl lipidadjuvant (GLA), and a pharmaceutically acceptable carrier that comprisesat least one of an oil and ISCOMATRIX™, and thereby altering hostimmunological responsiveness. In certain other embodiments there isprovided a method of altering immunological responsiveness in a host,comprising: administering to the host an immunological adjuvantcomposition that comprises a glycopyranosyl lipid adjuvant (GLA), andone or more of: (i) at least one co-adjuvant, (ii) at least one TLRagonist, (iii) at least one imidazoquinoline immune response modifier,and (iv) at least one double stem loop immune modifier (dSLIM), andthereby altering host immunological responsiveness.

In certain further embodiments, the co-adjuvant, when present, isselected from alum, a plant alkaloid and a detergent, wherein the plantalkaloid is tomatine and the detergent is selected from saponin,Polysorbate 80, Span 85 and Stearyl tyrosine, the TLR agonist, whenpresent, is selected from lipopolysaccharide, peptidoglycan, polyl:C,CpG, 3M003, flagellin, Leishmania homolog of eukaryotic ribosomalelongation and initiation factor 4a (LeIF) and at least one hepatitis Cantigen, and the imidazoquinoline immune response modifier, whenpresent, is selected from the group consisting of resiquimod (R848),imiquimod and gardiquimod.

In certain other embodiments there is provided a method of alteringimmunological responsiveness in a host, comprising: administering to thehost an immunological adjuvant composition that comprises aglycopyranosyl lipid adjuvant (GLA); and at least one of a co-adjuvantand a pharmaceutically acceptable carrier, wherein: the co-adjuvant isselected from the group consisting of a cytokine, a block copolymer orbiodegradable polymer, and a detergent, and the pharmaceuticallyacceptable carrier comprises a carrier that is selected from the groupconsisting of calcium phosphate, an oil-in-water emulsion, awater-in-oil emulsion, a liposome, and a microparticle, and therebyaltering host immunological responsiveness. In certain furtherembodiments the cytokine is selected from GM-CSF, IL-2, IL-7, IL-12,TNF-α and IFN-gamma, the block copolymer or biodegradable polymer isselected from Pluronic L121, CRL1005, PLGA, PLA, PLG, and polyl:C, andthe detergent is selected from the group consisting of saponin,Polysorbate 80, Span 85 and Stearyl tyrosine.

In certain further embodiments of the above described methods ofaltering immunological responsiveness in a host, the step ofadministering is performed one, two, three, four or more times. Incertain other further embodiments of the above described methods ofaltering immunological responsiveness in a host, altering immunologicalresponsiveness in the host comprises inducing or enhancing an immuneresponse. In certain other further embodiments of the above describedmethods of altering immunological responsiveness in a host, alteringimmunological responsiveness in the host comprises down-regulating animmune response. In certain further embodiments of the above describedmethods of altering immunological responsiveness in a host, the methodfurther comprises administering simultaneously or sequentially and ineither order an antigen that is derived from, or is immunologicallycross-reactive with, at least one infectious pathogen that is associatedwith an infectious disease against which induced or enhancedimmunological responsiveness is desired. In certain further suchembodiments the step of administering the antigen is performed one, two,three, four or more times. In certain other further embodiments of theabove described methods of altering immunological responsiveness in ahost, the method comprises administering simultaneously or sequentiallyand in either order an antigen that is derived from, or isimmunologically cross-reactive with, at least one epitope, biomolecule,cell or tissue that is associated with an autoimmune disease and againstwhich down-regulated immunological responsiveness is desired. In certainfurther such embodiments the step of administering the antigen isperformed one, two, three, four or more times. In certain other furtherembodiments of the above described methods of altering immunologicalresponsiveness in a host, the method comprises administeringsimultaneously or sequentially and in either order an antigen that isderived from, or is immunologically cross-reactive with, at least oneepitope, biomolecule, cell or tissue that is associated with a canceragainst which induced or enhanced immunological responsiveness isdesired. In certain further such embodiments the step of administeringthe antigen is performed one, two, three, four or more times.

In another embodiment there is provided a kit, comprising: animmunological adjuvant composition as described above in a firstcontainer; and an antigen in a second container, wherein theimmunological adjuvant composition is not in contact with the antigen.In another embodiment there is provided a kit, comprising: animmunological adjuvant composition as described above in a firstcontainer; and at least one recombinant expression construct whichcomprises a promoter operably linked to a nucleic acid sequence encodingan antigen, in a second container, wherein the immunological adjuvantcomposition is not in contact with the recombinant expression construct.In certain further embodiments of the just-described kit, the antigen isderived from at least one infectious pathogen that is selected from abacteria, a virus, a yeast and a protozoan. In certain other furtherembodiments of the just-described kit, the antigen is derived from atleast one cancer cell. In certain other further embodiments of thejust-described kit, the antigen is derived from, or is immunologicallycross-reactive with, at least one epitope, biomolecule, cell or tissuethat is associated with an autoimmune disease.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are set forth herein whichdescribe in more detail certain aspects of this invention, and aretherefore incorporated by reference in their entireties.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 shows HPLC data demonstrating the number and amounts ofcontaminating materials in MPL-AF and GLA-AF. These chromatograms werecollected using an Agilent 1100 system and an ESA Corona CAD detector.The method was run using a methanol to chloroform gradient on a WatersAtlantis C18 column. The injections included 2.5 μg of GLA and MPLrespectively and 0.27 μg of synthetic phosphocholine (POPC) which isused as a solubilizing agent.

FIG. 2 shows ELISA data demonstrating levels of cytokines and chemokinesexpressed by human macrophages of the Mono Mac 6 cell line (panels a-e),and PBMC-derived DC (panels f-h) in response to GLA stimulation. Cellswere cultured at 1×105 cells/well with an aqueous formulation of GSKBiologicals MPL® (MPL-AF), GLA (GLA-AF), or AF vehicle alone for 24 hrs.MIP-1b, IP-10, IL-6, IL-23 and IL-1b levels in supernatants weremeasured by sandwich ELISA.

FIG. 3 shows ELISA data demonstrating levels of anti-Fluzone antibodyproduction induced in mice one week after each immunization (i.e., atday 7, panel A; and at day 28, panel B) using two different doses ofFluzone vaccine formulated with GLA-AF, or GLA-SE, compared to Fluzonealone. Panels A & B show ELISA Ab titers of mice immunized twice at 3weeks interval with 20 ml (1.8 μg) or 2 ml (0.18 mg) of Fluzone (Flu)vaccine in a formulation containing GLA-AF, GLA-SE or no adjuvant, oneweek after the first (A) or second (B) injection. Panel C shows titersof neutralizing antibody (HAI) in the sera of mice after the secondimmunization.

FIG. 4 shows ELISA data demonstrating levels of anti-SMT antibodyproduction induced in mice one week after the third immunization usingSMT antigen alone, or formulated with GLA-SE. C57BL/6 mice wereimmunized three times at three-week intervals with SMT antigen (10 μgper animal for each immunization) formulated in a stable emulsioncontaining GLA (GLA-SE; 20 μg per animal for each immunization), orinjected with SMT protein alone. Sera were collected by bleeding oneweek after each immunization, and serum levels of IgG1, and IgG2cantibodies specific for SMT were examined by ELISA. Means and SEM ofreciprocal endpoint titers are shown.

FIG. 5 shows ELISA data demonstrating levels of anti-Leish-110f antibodyproduction induced in mice one week after the first immunization usingLeish-110f antigen formulated with different amounts of GLA (40, 20, 5,or 1 μg), compared to saline controls. Balb/c mice were immunized threetimes at two-week intervals with the Leish-110f antigen (10 μg peranimal for each immunization) formulated in a stable emulsion containing40, 20, 5, or 1 mg of GLA (GLA-SE), or injected with saline. Sera werecollected by bleeding one week after each immunization, and serum levelsof IgG1 and IgG2a antibodies specific for Leish-110f were examined byELISA. Means and SEM of reciprocal endpoint titers are shown for thesera collected 7 days after the 1st immunization.

FIG. 6 shows ELISA data demonstrating levels of anti-Leish-110f IFN-γcytokine production induced in mice one week after the thirdimmunization using Leish-110f antigen formulated with different amountsof GLA, compared to saline controls. Splenocytes, from Balb/c miceimmunized three times at two-week intervals with Leish-110f antigen (10μg) formulated in a stable emulsion containing 40, 5, or 1 μg of MPL(MPL-SE) or GLA (GLA-SE;), or from mice injected with a saline solution,were cultured for 3 days in vitro in medium alone, or in mediumcontaining 10 mg/ml of Leish-110f, or 3 mg/ml of Concanavalin A (ConA).IFN-g levels in supernatants were measured by ELISA. Means and SEM areshown.

FIG. 7 shows ICS data demonstrating the frequencies of ID83-specificIFN-γ, IL-2, and TNF cytokine producing CD4+ and CD8+ T cells induced inmice one week after the third immunization using ID83 alone oradjuvanted with formulations containing GLA (GLA-SE), GLA+CpG(GLA/CpG-SE), or GLA+GDQ (GLA/GDQ-SE). Splenocytes from C57BL/6 mice,immunized three times at three-week intervals with M. tuberculosis ID83fusion protein (8 μg) formulated with GLA-SE, GLA/CpG-SE,GLA/Gardiquimod (GDQ)-SE, or injected with saline, were cultured invitro for 12 hrs in medium containing 10 mg/ml of ID83. Cell levels ofIL-2, TNF, and IFN-g in CD3+CD4+ or CD3+CD8+ gated T cells were detectedby intracellular staining and measured by flow cytometry on a BD LSRIIFACS.

FIG. 8, panel A shows ICS data demonstrating the frequencies ofML0276-specific IFN-γ cytokine producing CD4+ T cells induced in miceone week after the third immunization using ML0276 antigen formulatedwith aqueous formulations containing CpG, or Imiquimod (IMQ), or astable oil emulsion containing GLA (GLA-SE), or the three mixedtogether, compared to saline and naïve controls. Splenocytes fromC57BL/6 mice, immunized three times at three-week intervals with M.leprea ML0276 antigen (10 μg) formulated with CpG, Imiquimod (IMQ),GLA-SE, a combination of the three, or injected with saline, werecultured for 12 hrs in vitro in medium containing 10 mg/ml of ML0276.Panel A shows cell levels of IFN-g in CD3+CD4+ T cells were detected byintracellular staining and measured by flow cytometry on a BD LSRIIFACS. Panel B shows draining lymph node cellularity as a correlate ofprotection.

FIG. 9 shows the surface expression of CD86 upon stimulation with GLA.Donor N003 CD14+ monocytes-derived primary dendritic cells wereincubated for 44 hours with 10,000 ng/ml, 1000 ng/ml, 100 ng/ml, 10ng/ml, 1 ng/ml, 0.1 ng/ml, or 0.01 ng/ml GLA (panel A) or MPL (panel B).A cytokine cocktail made of PGE₂, IL-1β, TNFα, and IL-6 was run as apositive control. Expression levels of the costimulatory molecule CD86at the surface of DC were used as an indicator of cell activation andmeasured by flow cytometry on a LSRII instrument (BD Biosciences, SanJose, Calif.) using CD86-specific fluorochrome—labeled antibody(eBiosciences, San Diego, Calif.).

FIG. 10 shows cultured human dendritic cells (DC) from three donors thatshow increased maturation with GLA stimulation. PBMC from three normaldonors was purified for human CD14+ monocytes-derived primary dendriticcells and stimulated with GLA (panels A-C) or MPL (panels D-F). Nostimulation was used as a negative control and a standard cytokinematuration cocktail of PGE2, IL-1β, TNFα, and IL-6 was run as a positivecontrol. The percent maximum expression of CD86-specificfluorochrome-labeled antibody (eBiosciences, San Diego, Calif.) was usedto monitor DC maturation by flow cytometry on a LSRII instrument (BDBiosciences, San Jose, Calif.)

FIG. 11 shows the lesion development in mice vaccinated withLeish-110f+GLA-SE upon challenge with L. major. Four Balb/c mice pertreatment group were immunized three times at two-week intervals witheither saline or the Leish-110f antigen (10 μg per animal for eachimmunization) formulated in a stable emulsion containing 20 μg of (i)GLA (Avanti Polar Lipids, Inc., Alabaster, Ala.; product number 699800;GLA-SE), or (ii) MPL® in an emulsion as supplied by the manufacturer(“MPL-SE”, GSK Biologicals, Rixensart, Belgium). Three weeks after thelast injection, mice were challenged intradermally in the pinea of bothears with 2×10³ purified Leishmania major clone V1 (MOHM/IL/80/Friedlin)metacyclic promastigotes. Development of cutaneous lesions was monitoredweekly for 6 weeks post-infection.

FIG. 12 shows parasite burden in mice vaccinated with Leish-110f

+GLA-SE upon challenge with L. major. Four Balb/c mice per treatmentgroup were immunized three times at two-week intervals with eithersaline or the Leish-110f antigen (10 μg per animal for eachimmunization) formulated in a stable emulsion containing 20 μg of (i)GLA (Avanti Polar Lipids, Inc., Alabaster, Ala.; product number 699800;GLA-SE), or (ii) MPL® in an emulsion as supplied by the manufacturer(“MPL-SE”, GSK Biologicals, Rixensart, Belgium). Three weeks after thelast injection, mice were challenged intradermally in the pinea of bothears with 2×10³ purified Leishmania major clone V1 (MOHM/IL/80/Friedlin)metacyclic promastigotes. Parasite burden in the ear and draining lymphnodes of infected mice were determined 6 weeks post-infection.Individual counts and mean are shown for each group. Student's t testwas performed and differences between groups were consideredstatistically significant when p<0.05.

FIG. 13 shows a flow cytometry analysis of OVA-specific T-cells.Single-cell analysis of OVA-specific CD8+ and CD4+ T cells producingsingle, double or triple Th1-type cytokines are shown. IFN-γ, IL-2 andTNF-α production by CD8+ (panels A-C) and CD4+ (panels D-F) T cells inresponse to in vitro stimulation with medium, P/I or OVA were evaluatedby flow cytometry. Splenocytes were purified from mice that wereinjected with saline, lentiviral vaccine or lentiviral vaccine plusGLA-SE, and were incubated in the presence of anti-CD28 and anti-CD49dwith the addition of medium or OVA.

FIG. 14 shows anti-Fluzone IgG antibody levels seven days following aboost immunization with Fluzone (two doses; 2 μg and 0.2 μg) givenintradermally (i.d.) with and without adjuvant. Panel A showsFluzone-specific IgG antibody responses; panel B shows Fluzone-specificIgG2a responses; panel C shows Fluzone-specific IgG1 responses; andpanel D shows IgG1:IgG2a ratios (results <1.0 represent an IgG2adominant response; results >1.0 represent an IgG1 dominant response).Asterisks represent statistical significance, p<0.05.

FIGS. 15A-D show cytokine levels for IFN-γ (15A), IL-10 (15B), IL-2(15C), and IL-5 (15D) from ex vivo stimulated splenocytes 3 weeksfollowing an intramuscular (i.m.) boost immunization with Fluzone (0.2μg) plus GLA-SE (5 μg). The average cytokine levels from threeindividual Balb/c mice per treatment group±s.d. are shown.

DETAILED DESCRIPTION OF THE INVENTION

The present invention in its several embodiments provides vaccinecompositions, adjuvant compositions, and related methods that includethe use of a synthetic glucopyranosyl lipid adjuvant (GLA). GLA providesa synthetic immunological adjuvant which, advantageously relative toadjuvants of the prior art, and in particular, relative to naturalproduct adjuvants, can be prepared in substantially homogeneous form.Moreover, GLA can be prepared efficiently and economically throughlarge-scale synthetic chemical manufacturing, unlike naturalproduct-derived adjuvants. As a synthetic adjuvant that is chemicallysynthesized from defined starting materials to obtain a chemicallydefined product that exhibits qualitative and quantitativebatch-to-batch consistency, GLA thus offers unprecedented benefitsincluding improved product quality control. Surprisingly, although3-acylated monophosphorylated lipid A has been associated with certaintoxicities, it has been found that when the 2 amine position contains asingle acyl chain, the molecules retain acceptable safety profiles.Further, the synthesis of such compounds is simplified because specificdeacylation at the 3 position presents technical challenges. Thus, theinvention offers further advantages in terms of safety and ease ofsynthesis.

As described herein, GLA-containing compositions and methods for theiruse include in some embodiments the use of GLA by itself with apharmaceutically acceptable carrier or excipient for immunologicaladjuvant activity, including “adjuvanting” in which GLA administrationto a subject may be wholly independent of, and/or separated temporallyand/or spatially from, administration to the subject of one or moreantigens against which elicitation or enhancement of an immune response(e.g., an antigen-specific response) in the subject is desired. Otherembodiments include the use of GLA in a vaccine composition that alsoincludes one or a plurality of antigens to which an immune responseelicited or enhanced by such a vaccine is desired. As described herein,these vaccine compositions may in certain related embodiments alsoinclude one or more toll-like receptor (TLR) agonist and/or one or aplurality of one or more of a co-adjuvant, an imidazoquinoline immuneresponse modifier, and a double stem loop immune modifier (dSLIM). Inother related embodiments, a vaccine composition as provided herein maycomprise GLA and one or more recombinant expression constructs eachcomprising a promoter operably linked to a nucleic acid sequenceencoding the antigen against which elicitation or enhancement of animmune response (e.g., an antigen-specific response) in the subject isdesired.

GLA

As also noted above, as a chemically synthesized adjuvant GLA can beprepared in substantially homogeneous form, which refers to a GLApreparation that is at least 80%, preferably at least 85%, morepreferably at least 90%, more preferably at least 95% and still morepreferably at least 96%, 97%, 98% or 99% pure with respect to the GLAmolecule.

In certain embodiments, GLA comprises (i) a diglucosamine backbonehaving a reducing terminus glucosamine linked to a non-reducing terminusglucosamine through an ether linkage between hexosamine position 1 ofthe non-reducing terminus glucosamine and hexosamine position 6 of thereducing terminus glucosamine; (ii) an O-phosphoryl group attached tohexosamine position 4 of the non-reducing terminus glucosamine; and(iii) up to six fatty acyl chains; wherein one of the fatty acyl chainsis attached to 3-hydroxy of the reducing terminus glucosamine through anester linkage, wherein one of the fatty acyl chains is attached to a2-amino of the non-reducing terminus glucosamine through an amidelinkage and comprises a tetradecanoyl chain linked to an alkanoyl chainof greater than 12 carbon atoms through an ester linkage, and whereinone of the fatty acyl chains is attached to 3-hydroxy of thenon-reducing terminus glucosamine through an ester linkage and comprisesa tetradecanoyl chain linked to an alkanoyl chain of greater than 12carbon atoms through an ester linkage. Determination of the degree ofpurity of a given GLA preparation can be readily made by those familiarwith the appropriate analytical chemistry methodologies, such as by gaschromatography, liquid chromatography, mass spectroscopy and/or nuclearmagnetic resonance analysis.

In certain embodiments, a GLA as used herein may have the followinggeneral structural formula:

where R¹, R³, R⁵ and R⁶ are C₁₁-C₂₀ alkyl; and R² and R⁴ are C₁₂-C₂₀alkyl.

In a more specific embodiment, the GLA has the formula set forth abovewherein R¹, R³, R⁵ and R⁶ are C₁₁₋₁₄ alkyl; and R² and R⁴ are C₁₂₋₁₅alkyl.

In a more specific embodiment, the GLA has the formula set forth abovewherein R¹, R³, R⁵ and R⁶ are C₁₁ alkyl; and R² and R⁴ are C₁₃ alkyl.

As demonstrated herein, it has unexpectedly been found that GLA hassurprisingly superior immunostimulatory activity when compared to MPL,while maintaining similar or reduced toxicity. For example, in certainembodiments, the GLA is effective for inducing an immune response thatis at least 2-fold, at least 3-fold, at least 5-fold or at least 10-foldmore potent than is induced using MPL at substantially the same orsimilar concentration. In other specific embodiments, the GLA hassubstantially the same or similar activity as MPL at concentrations atleast 5-fold, at least 10-fold, at least 25-fold or at least 100-foldlower than MPL.

Immune responses may be measured using any of a variety of knownimmunological assays or parameters known in the art and/or describedherein. For example, immune responses may be detected by any of avariety of well known parameters, including but not limited to in vivoor in vitro determination of: soluble immunoglobulins or antibodies;soluble mediators such as cytokines, lymphokines, chemokines, hormones,growth factors and the like as well as other soluble small peptide,carbohydrate, nucleotide and/or lipid mediators; cellular activationstate changes as determined by altered functional or structuralproperties of cells of the immune system, for example cellproliferation, altered motility, induction of specialized activitiessuch as specific gene expression or cytolytic behavior; cellulardifferentiation by cells of the immune system, including altered surfaceantigen expression profiles or the onset of apoptosis (programmed celldeath); or any other criterion by which the presence of an immuneresponse may be detected. In a specific embodiment, an immune responseis detected by measuring the induction of soluble mediators such ascytokines and/or chemokines (e.g., IFN-γ, IL-2, TNF, IL-1β, etc.). Inanother particular embodiment, an immune response may be detected bymeasuring in vivo protection from disease in an appropriate animalmodel.

GLA can be obtained commercially, for example, from Avanti Polar Lipids,Inc. (Alabaster, Ala.; product number 699800, wherein where R¹, R³, R⁵and R⁶ are undecyl and R² and R⁴ are dodecyl).

“Alkyl” means a straight chain or branched, noncyclic or cyclic,unsaturated or saturated aliphatic hydrocarbon containing from 1 to 20carbon atoms, and in certain preferred embodiments containing from 11 to20 carbon atoms. Representative saturated straight chain alkyls includemethyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like,including undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl,heptadecyl, octadecyl, etc.; while saturated branched alkyls includeisopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkylsinclude cyclopentenyl and cyclohexenyl, and the like. Cyclic alkyls arealso referred to herein as “homocycles” or “homocyclic rings.”Unsaturated alkyls contain at least one double or triple bond betweenadjacent carbon atoms (referred to as an “alkenyl” or “alkynyl”,respectively). Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like; while representative straightchain and branched alkynyls include acetylenyl, propynyl, 1-butynyl,2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.

Accordingly, in certain embodiments contemplated herein GLA may have anyof the above described structures, and in certain embodiments it isexpressly contemplated that GLA may, and in certain other embodiments itis expressly contemplated that GLA may not, have any structure of alipid adjuvant that is disclosed in one or more of U.S. Pat. No.6,544,518, EP 1531158, WO 2001/036433, WO 97/11708, WO 95/14026, U.S.Pat. No. 4,987,237, JP 63010728, JP 07055906, WO 2000/013029, U.S. Pat.No. 5,530,113, U.S. Pat. No. 5,612,476, U.S. Pat. No. 5,756,718, U.S.Pat. No. 5,843,918, WO 96/09310, U.S. Pub. 2004/161776, U.S. Pub. No.2003/170249, U.S. Pub. No. 2002/176867, WO 2002/032450, WO 2002/028424,WO 2002/016560, WO 2000/042994, WO 2000/025815, WO 2000?018929, JP10131046, WO 93/12778, EP 324455, DE 3833319, U.S. Pat. No. 4,844,894,U.S. Pat. No. 4,629,722. According to certain embodiments GLA is not3′-de-O-acylated.

Antigen

An antigen, for use in certain embodiments of the herein describedvaccine compositions and methods employing GLA, may be any targetepitope, molecule (including a biomolecule), molecular complex(including molecular complexes that contain biomolecules), subcellularassembly, cell or tissue against which elicitation or enhancement ofimmunreactivity in a subject is desired. Frequently, the term antigenwill refer to a polypeptide antigen of interest. However, antigen, asused herein, may also refer to a recombinant construct which encodes apolypeptide antigen of interest (e.g, an expression construct). Incertain preferred embodiments the antigen may be, or may be derivedfrom, or may be immunologically cross-reactive with, an infectiouspathogen and/or an epitope, biomolecule, cell or tissue that isassociated with infection, cancer, autoimmune disease, allergy, asthma,or any other condition where stimulation of an antigen-specific immuneresponse would be desirable or beneficial.

Preferably and in certain embodiments the vaccine formulations of thepresent invention contain an antigen or antigenic composition capable ofeliciting an immune response against a human or other mammalianpathogen, which antigen or antigenic composition may include acomposition derived from a virus such as from HIV-1, (such as tat, nef,gp120 or gp160), human herpes viruses, such as gD or derivatives thereofor Immediate Early protein such as ICP27 from HSV1 or HSV2,cytomegalovirus ((esp. Human)(such as gB or derivatives thereof),Rotavirus (including live-attenuated viruses), Epstein Barr virus (suchas gp350 or derivatives thereof), Varicella Zoster Virus (such as gpI,II and IE63), or from a hepatitis virus such as hepatitis B virus (forexample Hepatitis B Surface antigen or a derivative thereof), hepatitisA virus, hepatitis C virus and hepatitis E virus, or from other viralpathogens, such as paramyxoviruses: Respiratory Syncytial virus (such asF and G proteins or derivatives thereof), parainfluenza virus, measlesvirus, mumps virus, human papilloma viruses (for example HPV6, 11, 16,18, etc.), flaviviruses (e.g., Yellow Fever Virus, Dengue Virus,Tick-borne encephalitis virus, Japanese Encephalitis Virus) or Influenzavirus (whole live or inactivated virus, split influenza virus, grown ineggs or MDCK cells, or whole flu virosomes (as described by Gluck,Vaccine, 1992, 10, 915-920) or purified or recombinant proteins thereof,such as HA, NP, NA, or M proteins, or combinations thereof).

In certain other preferred embodiments the vaccine formulations of thepresent invention contain an antigen or antigenic composition capable ofeliciting an immune response against a human or other mammlian pathogen,which antigen or antigenic composition may include a composition derivedfrom one or more bacterial pathogens such as Neisseria spp, including N.gonorrhea and N. meningitidis (for example capsular polysaccharides andconjugates thereof, transferrin-binding proteins, lactoferrin bindingproteins, PiIC, adhesins); S. pyogenes (for example M proteins orfragments thereof, C5A protease, lipoteichoic acids), S. agalactiae, S.mutans: H. ducreyi; Moraxella spp, including M. catarrhalis, also knownas Branhamella catarrhalis (for example high and low molecular weightadhesins and invasins); Bordetella spp, including B. pertussis (forexample pertactin, pertussis toxin or derivatives thereof, filamenteoushemagglutinin, adenylate cyclase, fimbriae), B. parapertussis and B.bronchiseptica; Mycobacterium spp., including M. tuberculosis (forexample ESAT6, Antigen 85A, —B or —C), M. bovis, M. leprae, M. avium, M.paratuberculosis, M. smegmatis; Legionella spp, including L.pneumophila; Escherichia spp, including enterotoxic E. coli (for examplecolonization factors, heat-labile toxin or derivatives thereof,heat-stable toxin or derivatives thereof), enterohemorragic E. coli,enteropathogenic E. coli (for example shiga toxin-like toxin orderivatives thereof); Vibrio spp, including V. cholera (for examplecholera toxin or derivatives thereof); Shigella spp, including S.sonnei, S. dysenteriae, S. flexnerii; Yersinia spp, including Y.enterocolitica (for example a Yop protein), Y. pestis, Y.pseudotuberculosis; Campylobacter spp, including C. jejuni (for exampletoxins, adhesins and invasins) and C. coli; Salmonella spp, including S.typhi, S. paratyphi, S. choleraesuis, S. enteritidis; Listeria spp.,including L. monocytogenes; Helicobacter spp, including H. pylori (forexample urease, catalase, vacuolating toxin); Pseudomonas spp, includingP. aeruginosa; Staphylococcus spp., including S. aureus, S. epidermidis;Enterococcus spp., including E. faecalis, E. faecium; Clostridium spp.,including C. tetani (for example tetanus toxin and derivative thereof),C. botulinum (for example botulinum toxin and derivative thereof), C.difficile (for example clostridium toxins A or B and derivativesthereof); Bacillus spp., including B. anthracis (for example botulinumtoxin and derivatives thereof); Corynebacterium spp., including C.diphtheriae (for example diphtheria toxin and derivatives thereof);Borrelia spp., including B. burgdorferi (for example OspA, OspC, DbpA,DbpB), B. garinii (for example OspA, OspC, DbpA, DbpB), B. afzelii (forexample OspA, OspC, DbpA, DbpB), B. andersonii (for example OspA, OspC,DbpA, DbpB), B. hermsii; Ehrlichia spp., including E. equi and the agentof the Human Granulocytic Ehrlichiosis; Rickettsia spp, including R.rickettsii; Chlamydia spp. including C. trachomatis (for example MOMP,heparin-binding proteins), C. pneumoniae (for example MOMP,heparin-binding proteins), C. psittaci; Leptospira spp., including L.interrogans; Treponema spp., including T. pallidum (for example the rareouter membrane proteins), T. denticola, T. hyodysenteriae; or otherbacterial pathogens.

In certain other preferred embodiments the vaccine formulations of thepresent invention contain an antigen or antigenic composition capable ofeliciting an immune response against a human or other mammalianpathogen, which antigen or antigenic composition may include acompostion derived from one or more parasites (See, e.g., John, D. T.and Petri, W. A., Markell and Voge's Medical Parasitology-9^(th) Ed.,2006, WB Saunders, Philadelphia; Bowman, D. D., Georgis' Parasitologyfor Veterinarians-8^(th) Ed., 2002, WB Saunders, Philadelphia) such asPlasmodium spp., including P. falciparum; Toxoplasma spp., including T.gondii (for example SAG2, SAG3, Tg34); Entamoeba spp., including E.histolytica; Babesia spp., including B. microti; Trypanosoma spp.,including T. cruzi; Giardia spp., including G. lamblia; Leshmania spp.,including L. major; Pneumocystis spp., including P. carinii; Trichomonasspp., including T. vaginalis; or from a helminth capable of infecting amammal, such as: (i) nematode infections (including, but not limited to,Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichuria,Necator americanus, Ancylostoma duodenale, Wuchereria bancrofti, Brugiamalayi, Onchocerca volvulus, Dracanculus medinensis, Trichinellaspiralis, and Strongyloides stercoralis); (ii) trematode infections(including, but not limited to, Schistosoma mansoni, Schistosomahaematobium, Schistosoma japonicum, Schistosoma mekongi, Opisthorchissinensis, Paragonimus sp, Fasciola hepatica, Fasciola magna, Fasciolagigantica); and (iii) cestode infections (including, but not limited to,Taenia saginata and Taenia solium). Certain embodiments may thereforecontemplate vaccine compositions that include an antigen derived fromSchisostoma spp., Schistosoma mansonii, Schistosoma haematobium, and/orSchistosoma japonicum, or derived from yeast such as Candida spp.,including C. albicans; Cryptococcus spp., including C. neoformans.

Other preferred specific antigens for M. tuberculosis are for example ThRa12, Tb H9, Tb Ra35, Tb38-1, Erd 14, DPV, MTI, MSL, mTTC2 and hTCC1 (WO99/51748). Proteins for M. tuberculosis also include fusion proteins andvariants thereof where at least two, preferably three polypeptides of M.tuberculosis are fused into a larger protein. Preferred fusions includeRa12-TbH9-Ra35, Erd14-DPV-MTI, DPV-MTI-MSL, Erd14DPV-MTI-MSL-mTCC2,Erd14-DPV-MTI-MSL, DPV-MTI-MSL-mTCC2, TbH9-DPV-MTI (WO 99151748).

Most preferred antigens for Chlamydia include for example the HighMolecular Weight Protein (HWMP) (WO 99/17741), ORF3 (EP 366 412), andputative membrane proteins (Pmps). Other Chlamydia antigens of thevaccine formulation can be selected from the group described in WO99128475. Preferred bacterial vaccines comprise antigens derived fromStreptococcus spp, including S. pneumoniae (for example capsularpolysaccharides and conjugates thereof, PsaA, PspA, streptolysin,choline-binding proteins) and the protein antigen Pneumolysin (BiochemBiophys Acta, 1989, 67, 1007; Rubins et al., Microbial Pathogenesis, 25,337-342), and mutant detoxified derivatives thereof (WO 90/06951; WO99/03884). Other preferred bacterial vaccines comprise antigens derivedfrom Haemophilus spp., including H. influenzae type B (for example PRPand conjugates thereof), non typeable H. influenzae, for example OMP26,high molecular weight adhesins, P5, P6, protein D and lipoprotein D, andfimbrin and fimbrin derived peptides (U.S. Pat. No. 5,843,464) ormultiple copy varients or fusion proteins thereof.

Derivatives of Hepatitis B Surface antigen are well known in the art andinclude, inter alia, those PreS1, Pars2 S antigens set forth describedin European Patent applications EP-A414 374; EP-A-0304 578, and EP198474. In one preferred aspect the vaccine formulation of the inventioncomprises the HIV-1 antigen, gp120, especially when expressed in CHOcells. In a further embodiment, the vaccine formulation of the inventioncomprises gD2t as hereinabove defined.

In a preferred embodiment of the present invention vaccines containingthe claimed adjuvant comprise antigen derived from the Human PapillomaVirus (HPV) considered to be responsible for genital warts (HPV 6 or HPV11 and others), and the HPV viruses responsible for cervical cancer(HPV16, HPV18 and others). Particularly preferred forms of genital wartprophylactic, or therapeutic, vaccine comprise L1 particles orcapsomers, and fusion proteins comprising one or more antigens selectedfrom the HPV 6 and HPV 11 proteins E6, E7, L1, and L2. Certain preferredforms of fusion protein include L2E7 as disclosed in WO 96/26277, andproteinD(1/3)-E7 disclosed in GB 9717953.5 (PCT/EP98/05285). A preferredHPV cervical infection or cancer, prophylaxis or therapeutic vaccine,composition may comprise HPV 16 or 18 antigens. For example, L1 or L2antigen monomers, or L1 or L2 antigens presented together as a viruslike particle (VLP) or the L1 alone protein presented alone in a VLP orcaposmer structure. Such antigens, virus like particles and capsomer areper se known. See for example WO94/00152, WO94/20137, WO94/05792, andWO93/02184.

Additional early proteins may be included alone or as fusion proteinssuch as E7, E2 or preferably F5 for example; particularly preferredembodiments include a VLP comprising L1 E7 fusion proteins (WO96/11272). Particularly preferred HPV 16 antigens comprise the earlyproteins E6 or F7 in fusion with a protein D carrier to form ProteinD-E6 or E7 fusions from HPV 16, or combinations thereof; or combinationsof E6 or E7 with L2 (WO 96/26277). Alternatively the HPV 16 or 18 earlyproteins E6 and E7, may be presented in a single molecule, preferably aProtein D-E6/E7 fusion. Such vaccine may optionally contain either orboth E6 and E7 proteins front HPV 18, preferably in the form of aProtein D-E6 or Protein D-E7 fusion protein or Protein D E6/E7 fusionprotein. The vaccine of the present invention may additionally compriseantigens from other HPV strains, preferably from strains HPV 31 or 33.

Vaccines of the present invention further comprise antigens derived fromparasites that cause Malaria. For example, preferred antigens fromPlasmodia falciparum include RTS,S and TRAP. RTS is a hybrid proteincomprising substantially all the C-terminal portion of thecircumsporozoite (CS) protein of P. falciparum linked via four aminoacids of the preS2 portion of Hepatitis B surface antigen to the surface(S) antigen of hepatitis B virus. Its full structure is disclosed in theInternational Patent Application No. PCT/EP92/02591, published as WO93/10152 claiming priority from UK patent application No. 9124390.7.When expressed in yeast RTS is produced as a lipoprotein particle, andwhen it is co-expressed with the S antigen from HBV it produces a mixedparticle known as RTS,S.

TRAP antigens are described in the International Patent Application No.PCT/GB89/00895 published as WO 90/01496. A preferred embodiment of thepresent invention is a Malaria vaccine wherein the antigenic preparationcomprises a combination of the RTS,S and TRAP antigens. Other plasmodiaantigens that are likely candidates to be components of a multistageMalaria vaccine are P. faciparum MSP1, AMA1, MSP3, EBA, GLURP, RAP1,RAP2, Sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP, SALSA, PfEXP1,Pfs25, Pfs28, PFS27125, Pfs16, Pfs48/45, Pfs230 and their analogues inPlasmodium spp.

Accordingly, certain herein disclosed embodiment contemplate an antigenthat is derived from at least one infectious pathogen such as abacterium, a virus or a fungus, including an Actinobacterium such as M.tuberculosis or M. leprae or another mycobacterium; a bacterium such asa member of the genus Salmonella, Neisseria, Borrelia, Chlamydia orBordetella; a virus such as a herpes simplex virus, a humanimmunodeficiency virus (HIV), a feline immunodeficiency virus (FIV),cytomegalovirus, Varicella Zoster Virus, hepatitis virus, Epstein BarrVirus (EBV), respiratory syncytial virus, human papilloma virus (HPV)and a cytomegalovirus; HIV such as HIV-1 or HIV-2; a fungus such asAspergillus, Blastomyces, Coccidioides and Pneumocysti or a yeast,including Candida species such as C. albicans, C. glabrata, C. krusei,C. lusitaniae, C. tropicalis and C. parapsilosis; a parasite such as aprotozoan, for example, a Plasmodium species including P. falciparum, P.vivax, P. malariae and P. ovale; or another parasite such as one or moreof Acanthamoeba, Entamoeba histolytica, Angiostrongylus, Schistosomamansonii, Schistosoma haematobium, Schistosoma japonicum,Cryptosporidium, Ancylostoma, Entamoeba histolytica, Entamoeba coli,Entamoeba dispar, Entamoeba hartmanni, Entamoeba polecki, Wuchereriabancrofti, Giardia, and Leishmania.

For example, in GLA-containing vaccine embodiments containing antigensderived from Borrelia sp., the antigens may include nucleic acid,pathogen derived antigen or antigenic preparations, recombinantlyproduced protein or peptides, and chimeric fusion proteins. One suchantigen is OspA. The OspA may be a full mature protein in a lipidatedform by virtue of its biosynthesis in a host cell (Lipo-OspA) or mayalternatively be a non-lipidated derivative. Such non-lipidatedderivatives include the non-lipidated NS1-OspA fusion protein which hasthe first 81 N-terminal amino acids of the non-structural protein (NS1)of the influenza virus, and the complete OspA protein, and another,MDP-OspA is a non-lipidated form of OspA carrying 3 additionalN-terminal amino acids.

Compositions and methods are known in the art for identifying subjectshaving, or suspected of being at risk for having, an infection with aninfectious pathogen as described herein.

For example, the bacterium Mycobacterium tuberculosis cases tuberculosis(TB). The bacteria usually attack the lungs but can also attack thekidney, spine, and brain. If not treated properly, TB disease can befatal. The disease is spread from one person to another in the air whenan infected person sneezes or coughs. In 2003, more than 14,000 cases ofTB were reported in the United States.

Although tuberculosis can generally be controlled using extendedantibiotic therapy, such treatment is not sufficient to prevent thespread of the disease and concerns exist regarding the potentialselection for antibiotic-resistant strains. Infected individuals may beasymptomatic, but contagious, for some time. In addition, althoughcompliance with the treatment regimen is critical, patient behavior isdifficult to monitor. Some patients do not complete the course oftreatment, which can lead to ineffective treatment and the developmentof drug resistance. (e.g., U.S. Pat. No. 7,087,713)

Currently, vaccination with live bacteria is the most efficient methodfor inducing protective immunity against tuberculosis. The most commonMycobacterium employed for this purpose is Bacillus Calmette-Guerin(BCG), an avirulent strain of Mycobacterium bovis. However, the safetyand efficacy of BCG is a source of controversy and some countries, suchas the United States, do not vaccinate the general public. Diagnosis iscommonly achieved using a skin test, which involves intradermal exposureto tuberculin PPD (protein-purified derivative). Antigen-specific T cellresponses result in measurable induration at the injection site by 48 72hours after injection, which indicates exposure to Mycobacterialantigens. Sensitivity and specificity have, however, been a problem withthis test, and individuals vaccinated with BCG cannot be distinguishedfrom infected individuals. (e.g., U.S. Pat. No. 7,087,713)

While macrophages have been shown to act as the principal effectors ofM. tuberculosis immunity, T cells are the predominant inducers of suchimmunity. The essential role of T cells in protection against M.tuberculosis infection is illustrated by the frequent occurrence of M.tuberculosis in AIDS patients, due to the depletion of CD4 T cellsassociated with human immunodeficiency virus (HIV) infection.Mycobacterium-reactive CD4 T cells have been shown to be potentproducers of gamma-interferon (IFN-gamma), which, in turn, has beenshown to trigger the anti-mycobacterial effects of macrophages in mice.While the role of IFN-gamma in humans is less clear, studies have shownthat 1,25-dihydroxy-vitamin D3, either alone or in combination withIFN-gamma or tumor necrosis factor-alpha, activates human macrophages toinhibit M. tuberculosis infection. Furthermore, it is known thatIFN-gamma stimulates human macrophages to make 1,25-dihydroxy-vitaminD3. Similarly, IL-12 has been shown to play a role in stimulatingresistance to M. tuberculosis infection. For a review of the immunologyof M. tuberculosis infection, see Chan and Kaufmann, in Tuberculosis:Pathogenesis, Protection and Control, Bloom (ed.), ASM Press.Washington, D.C. (1994).

Existing compounds and methods for diagnosing tuberculosis or forinducing protective immunity against tuberculosis include the use ofpolypeptides that contain at least one immunogenic portion of one ormore Mycobacterium proteins and DNA molecules encoding suchpolypeptides. Diagnostic kits containing such polypeptides or DNAsequences and a suitable detection reagent may be used for the detectionof Mycobacterium infection in patients and biological samples.Antibodies directed against such polypeptides are also provided. Inaddition, such compounds may be formulated into vaccines and/orpharmaceutical compositions for immunization against Mycobacteriuminfection. (U.S. Pat. Nos. 6,949,246 and 6,555,653).

Malaria was eliminated in many parts of the world in the 1960s, but thedisease still persists and new strains of the disease are emerging thatare resistant to existing drugs. Malaria is a major public healthproblem in more than 90 countries. Nine out of ten cases of malariaoccur in sub-Saharan Africa. More than one third of the world'spopulation is at risk, and between 350 and 500 million people areinfected with malaria each year. Forty-five million pregnant women areat risk of contracting malaria this year. Of those individuals alreadyinfected, more than 1 million of those infected die each year from whatis a preventable disease. The majority of those deaths are children inAfrica.

Malaria is usually transmitted when a person is bitten by an infectedfemale Anopheles mosquito. To transmit the mosquito must have beeninfected by having drawn blood from a person already infected withmalaria. Malaria is caused by a parasite and the clinical symptoms ofthe disease include fever and flu-like illness, such as chills,headache, muscle aches, and tiredness. These symptoms may be accompaniedby nausea, vomiting, and diarrhea. Malaria can also cause anemia andjaundice because of the loss of red blood cells. Infection with one typeof malaria, Plasmodium falciparum, if not promptly treated, may causekidney failure, seizures, mental confusion, coma, and death.

An in vitro diagnostic method for malaria in an individual is known,comprising placing a tissue or a biological fluid taken from anindividual in contact with a molecule or polypeptide composition,wherein said molecule or polypeptide composition comprises one or morepeptide sequences bearing all or part of one or more T epitopes of theproteins resulting from the infectious activity of P. falciparum, underconditions allowing an in vitro immunological reaction to occur betweensaid composition and the antibodies that may be present in the tissue orbiological fluid, and in vitro detection of the antigen-antibodycomplexes formed (see, e.g., U.S. Pat. No. 7,087,231). Expression andpurification of a recombinant Plasmodium falciparum (3D7) AMA-1ectodomain have been described. Previous methods have produced a highlypurified protein which retains folding and disulfide bridging of thenative molecule. The recombinant AMA-1 is useful as a diagnosticreagentas well as in antibody production, and as a protein for usealone, or as part of, a vaccine to prevent malaria. (U.S. Pat. No.7,029,685)

Polynucleotides have been described in the art that encodespecies-specific P. vivax malarial peptide antigens which are proteinsor fragments of proteins secreted into the plasma of a susceptiblemammalian host after infection, as have monoclonal or polyclonalantibodies directed against these antigens. The peptide antigens,monoclonal antibodies, and/or polyclonal antibodies are utilized inassays used to diagnose malaria, as well as to determine whetherPlasmodium vivax is the species responsible for the infection. (U.S.Pat. No. 6,706,872) Species-specific P. vivax malarial peptide antigenshave also been reported which are proteins or fragments of proteinssecreted into the plasma of a susceptible mammalian host afterinfection, as have monoclonal or polyclonal antibodies directed againstthese antigens. The peptide antigens, monoclonal antibodies, and/orpolyclonal antibodies are utilized in assays used to diagnose malaria,as well as to determine whether Plasmodium vivax is the speciesresponsible for the infection (see, e.g., U.S. Pat. No. 6,231,861).

A recombinant Plasmodium falciparum (3D7) AMA-1 ectodomain has also beenexpressed by a method that produces a highly purified protein whichretains folding and disulfide bridging of the native molecule. Therecombinant AMA-1 is useful as a diagnostic reagent, for use in antibodyproduction, and as a vaccine. (U.S. Pat. No. 7,060,276) Similarly knownare the expression and purification of a recombinant Plasmodiumfalciparum (3D7) MSP-1₄₂, which retains folding and disulfide bridgingof the native molecule. The recombinant MSP-1₄₂ is useful as adiagnostic reagent, for use in antibody production, and as a vaccine.(U.S. Pat. No. 6,855,322)

Diagnostic methods for the detection of human malaria infections toidentify a subject having or suspected of being at risk for having aninfection with a malaria infectious pathogen are thus known according tothese and related disclosures. Specifically, for example, blood samplesare combined with a reagent containing 3-acetyl pyridine adeninedinucleotide (APAD), a substrate (e.g. a lactate salt or lactic acid),and a buffer. The reagent is designed to detect the presence of a uniqueglycolytic enzyme produced by the malaria parasite. This enzyme is knownas parasite lactic acid dehydrogenase (PLDH). PLDH is readilydistinguishable from host LDH using the above-described reagent.Combination of the reagent with a parasitized blood sample results inthe reduction of APAD. However, APAD is not reduced by host LDH. Thereduced APAD may then be detected by various techniques, includingspectral, fluorimetric, electrophoretic, or colorimetric analysis.Detection of the reduced APAD in the foregoing manner provides apositive indication of malaria infection (e.g., U.S. Pat. No.5,124,141). In another methodology for diagnosing malaria, a polypeptidecomprising a characteristic amino acid sequence derived from thePlasmodium falciparum antigen GLURP, is recognized in a test sample by aspecific antibody raised against or reactive with the polypeptide. (U.S.Pat. No. 5,231,168)

Leishmaniasis is a widespread parasitic disease with frequent epidemicsin the Indian subcontinent, Africa, and Latin America and is a WorldHealth Organization priority for vaccine development. A complex ofdifferent diseases, Leishmania parasites cause fatal infections ofinternal organs, as well as serious skin disease. One of the mostdevastating forms of leishmaniasis is a disfiguring infection of thenose and mouth. The number of cases of leishmaniasis are increasing, andit is now out of control in many areas. Leishmaniasis is also on therise in some developed countries, specifically southern Europe as aresult of HIV infection. Available drugs are toxic, expensive, andrequire long-term daily injections.

Leishmania are protozoan parasites that inhabit macrophages or the whiteblood cells of the immune system. The parasites are transmitted by thebite of small blood sucking insects (sand flies), which are difficult tocontrol, as they inhabit vast areas of the planet.

Visceral leishmaniasis is the most dangerous of the three manifestationsof the disease. It is estimated that about 500,000 new cases of thevisceral form (kala-azar or “the killing disease”) occur each year. Morethan 200 million people are currently at risk for contracting visceralleishmaniasis. Over 90 percent of visceral leishmaniasis cases occur inIndia, Bangladesh, Sudan, Brazil, and Nepal. Most of the deaths occur inchildren. Those with the cutaneous forms are often left permanentlydisfigured.

Leishmania infections are difficult to diagnose and typically involvehistopathologic analysis of tissue biopsy specimens. Several serologicaland immunological diagnostic assays have, however, been developed. (U.S.Pat. No. 7,008,774; Senaldi et al., (1996)J. Immunol. Methods 193:9 5;Zijlstra, et al., (1997) Trans. R. Soc. Trop. Med. Hyg. 91:671 673;Badaro, et al., (1996) J. Inf. Dis. 173:758 761; Choudhary, S., et al.,(1992) J. Comm. Dis. 24:32 36; Badaro, R., et al., (1986) Am. J. Trop.Med. Hyg. 35:72 78; Choudhary, A., et al., (1990) Trans. R. Soc. Trop.Med. Hyg. 84:363 366; and Reed, S. G., et al., (1990) Am. J. Trop. Med.Hyg. 43:632 639). The promastigotes release metabolic products into theculture medium to produce conditioned medium. These metabolic productsare immunogenic to the host. See Schnur, L. F., et al., (1972) Isrl. J.Med. Sci. 8:932 942; Sergeiev, V. P., et al., (1969) Med. Parasitol.38:208 212; E1-On, J., et al., (1979) Exper. Parasitol. 47:254 269; andBray, R. S., et al., (1966) Trans. R. Soc. Trop. Med. Hyg. 60:605 609;U.S. Pat. No. 6,846,648, U.S. Pat. No. 5,912,166; U.S. Pat. No.5,719,263; U.S. Pat. No. 5,411,865).

About 40 million people around the world are infected with HIV, thevirus that causes AIDS. Around 3 million people die of the disease eachyear, 95 percent of them in the developing world. Each year, close to 5million people become infected with HIV. Currently, sub-Saharan Africancarries the highest burden of disease, but it is quickly spreading toother countries such as India, China, and Russia. The epidemic isgrowing most rapidly among minority populations. In the United Statesthere have been more than 950,000 cases of AIDS reported since 1981.AIDS hits people during their most productive years. Women, for bothbiological and social reasons, have an increased risk for HIV/AIDS.

AIDS is caused by human immunodeficiency virus (HIV), which kills anddamages cells of the body's immune system and progressively destroys thebody's ability to fight infections and certain cancers. HIV is spreadmost commonly by having unprotected sex with an infected partner. Themost robust solution to the problem is preventing the virus fromspreading. Making a safe, effective, and affordable HIV vaccine is oneway to reach this goal. Across the world, fewer than one in five peopleat high risk for HIV infection have access to effective prevention.

Methods for diagnosing HIV infections are known, including by virusculture, PCR of definitive nucleic acid sequences from patientspecimens, and antibody tests for the presence of anti-HIV antibodies inpatient sera, (see e.g., U.S. Pat. Nos. 6,979,535, 6,544,728, 6,316,183,6,261,762, 4,743,540.)

According to certain other embodiments as disclosed herein, the vaccinecompositions and related formulations and methods of use may include anantigen that is derived from a cancer cell, as may be useful for theimmunotherapeutic treatment of cancers. For example, the adjuvantformulation may finds utility with tumor rejection antigens such asthose for prostate, breast, colorectal, lung, pancreatic, renal ormelanoma cancers. Exemplary cancer or cancer cell-derived antigensinclude MAGE 1, 3 and MAGE 4 or other MAGE antigens such as thosedisclosed in WO99/40188, PRAME, BAGE, Lage (also known as NY Eos 1) SAGEand HAGE (WO 99/53061) or GAGE (Robbins and Kawakami, 1996 CurrentOpinions in Immunology 8, pps 628-636; Van den Eynde et al.,International Journal of Clinical & Laboratory Research (1997 & 1998);Correale et al. (1997), Journal of the National Cancer Institute 89, p.293. These non-limiting examples of cancer antigens are expressed in awide range of tumor types such as melanoma, lung carcinoma, sarcoma andbladder carcinoma. See, e.g., U.S. Pat. No. 6,544,518.

Other tumor-specific antigens are suitable for use with GLA according tocertain presently disclosed embodiments include, but are not restrictedto, tumor-specific or tumor-associated gangliosides such as GM₂, and GM₃or conjugates thereof to carrier proteins; or an antigen for use in aGLA vaccine composition for eliciting or enhancing an anti-cancer immuneresponse may be a self peptide hormone such as whole lengthGonadotrophin hormone releasing hormone (GnRH, WO 95/20600), a short 10amino acid long peptide, useful in the treatment of many cancers. Inanother embodiment prostate antigens are used, such as Prostate specificantigen (PSA), PAP, PSCA (e.g., Proc. Nat. Acad. Sci. USA 95(4)1735-1740 1998), PSMA or, in a preferred embodiment an antigen known asProstase. (e.g., Nelson, et al., Proc. Natl. Acad. Sci. USA(1999) 96:3114-3119; Ferguson, et al. Proc. Natl. Acad. Sci. USA 1999. 96,3114-3119; WO 98/12302; U.S. Pat. No. 5,955,306; WO 98/20117; U.S. Pat.Nos. 5,840,871 and 5,786,148; WO 00/04149. Other prostate specificantigens are known from WO 98/137418, and WO/004149. Another is STEAP(PNAS 96 14523 14528 7-12 1999).

Other tumor associated antigens useful in the context of the presentinvention include: Plu-1 (J. Biol. Chem. 274 (22) 15633-15645, 1999),HASH-1, HasH-2, Cripto (Salomon et al Bioessays 199, 21:61-70, U.S. Pat.No. 5,654,140) and Criptin (U.S. Pat. No. 5,981,215). Additionally,antigens particularly relevant for vaccines in the therapy of canceralso comprise tyrosinase and survivin.

The herein disclosed embodiments pertaining to GLA-containing vaccinecompositions comprising a cancer antigen will be useful against anycancer characterised by tumor associated antigen expression, such asHER-2/neu expression or other cancer-specific or cancer-associatedantigens.

Diagnosis of cancer in a subject having or suspected of being at riskfor having cancer may be accomplished by any of a wide range ofart-accepted methodologies, which may vary depending on a variety offactors including clinical presentation, degree of progression of thecancer, the type of cancer, and other factors. Examples of cancerdiagnostics include histopathological, histocytochemical,immunohistocytochemical and immunohistopathological examination ofpatient samples (e.g., blood, skin biopsy, other tissue biopsy, surgicalspecimens, etc.), PCR tests for defined genetic (e.g., nucleic acid)markers, serological tests for circulating cancer-associated antigens orcells bearing such antigens, or for antibodies of defined specificity,or other methodologies with which those skilled in the art will befamiliar. See, e.g., U.S. Pat. Nos. 6,734,172; 6,770,445; 6,893,820;6,979,730; 7,060,802; 7,030,232; 6,933,123; 6,682,901; 6,587,792;6,512,102; 7,078,180; 7,070,931; JP5-328975; Waslylyk et al., 1993 Eur.J. Bloch. 211(7):18.

Vaccine compositions and methods according to certain embodiments of thepresent invention may also be used for the prophylaxis or therapy ofautoimmune diseases, which include diseases, conditions or disorderswherein a host's or subject's immune system detrimentally mediates animmune response that is directed against “self” tissues, cells,biomolecules (e.g., peptides, polypeptides, proteins, glycoproteins,lipoproteins, proteolipids, lipids, glycolipids, nucleic acids such asRNA and DNA, oligosaccharides, polysaccharides, proteoglycans,glycosaminoglycans, or the like, and other molecular components of thesubjects cells and tissues) or epitopes (e.g., specific immunologicallydefined recognition structures such as those recognized by an antibodyvariable region complementarity determining region (CDR) or by a T cellreceptor CDR.

Autoimmune diseases are thus characterized by an abnormal immuneresponse involving either cells or antibodies, that are in either casedirected against normal autologous tissues. Autoimmune diseases inmammals can generally be classified in one of two different categories:cell-mediated disease (i.e., T-cell) or antibody-mediated disorders.Non-limiting examples of cell-mediated autoimmune diseases includemultiple sclerosis, rheumatoid arthritis, Hashimoto thyroiditis, type Idiabetes mellitus (Juvenile onset diabetes) and autoimmune uvoretinitis.Antibody-mediated autoimmune disorders include, but are not limited to,myasthenia gravis, systemic lupus erythematosus (or SLE), Graves'disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia,autoimmune asthma, cryoglobulinemia, thrombic thrombocytopenic purpura,primary biliary sclerosis and pernicious anemia. The antigen(s)associated with: systemic lupus erythematosus is small nuclearribonucleic acid proteins (snRNP); Graves' disease is the thyrotropinreceptor, thyroglobulin and other components of thyroid epithelial cells(Akamizu et al., 1996; Kellerman et al., 1995; Raju et al., 1997; andTexier et al., 1992); pemphigus is cadherin-like pemphigus antigens suchas desmoglein 3 and other adhesion molecules (Memar et al., 1996:Stanley, 1995; Plott et al., 1994; and Hashimoto, 1993); and thrombicthrombocytopenic purpura is antigens of platelets. (See, e.g., U.S. Pat.No. 6,929,796; Gorski et al. (Eds.), Autoimmunity, 2001, Kluwer AcademicPublishers, Norwell, M A; Radbruch and Lipsky, P. E. (Eds.) CurrentConcepts in Autoimmunity and Chronic Inflammation (Curr. Top. Microbiol.and Immunol.) 2001, Springer, N.Y.)

Autoimmunity plays a role in more than 80 different diseases, includingtype 1 diabetes, multiple sclerosis, lupus, rheumatoid arthritis,scleroderma, and thyroid diseases. Vigorous quantitative estimates ofmorbidity for most autoimmune diseases are lacking. Most recent studiesdone in the late 1990s reveal that autoimmune diseases are the thirdmost common major illness in the United States; and the most commonautoimmune diseases affect more than 8.5 million Americans. Currentestimates of the prevalence of the disease range from 5 to 8 percent ofthe United States population. Most autoimmune diseasesdisproportionately affect women. Women are 2.7 times more likely thanmen to acquire an autoimmune disease. Women are more susceptible toautoimmune diseases; men appear to have higher levels of natural killercell activity than do women. (Jacobsen et al, Clinical Immunology andImmunopathology, 84:223-243, 1997.)

Autoimmune diseases occur when the immune system mistakes self tissuesfor nonself and mounts an inappropriate attack. The body can be affectedin different ways from autoimmune diseases, including, for example, thegut (Crohn's disease) and the brain (multiple sclerosis). It is knownthat an autoantibody attacks self-cells or self-tissues to injure theirfunction and as a result causes autoimmune diseases, and that theautoantibody may be detected in the patient's serum prior to the actualoccurrence of an autoimmune disease (e.g., appearance of clinical signsand symptoms). Detection of an autoantibody thus permits early discoveryor recognition of presence or risk for developing an autoimmune disease.Based on these findings, a variety of autoantibodies againstautoantigens have been discovered and the autoantibodies againstautoantigens have been measured in clinical tests (e.g., U.S. Pat. Nos.6,919,210, 6,596,501, 7,012,134, 6,919,078) while other autoimmunediagnostics may involve detection of a relevant metabolite (e.g., U.S.Pat. No. 4,659,659) or immunological reactivity (e.g., U.S. Pat. Nos.4,614,722 and 5,147,785, 4,420,558, 5,298,396, 5,162,990, 4,420,461,4,595,654, 5,846,758, 6,660,487).

In certain embodiments, the compositions of the invention will beparticularly applicable in treatment of the elderly and/or theimmunosuppressed, including subjects on kidney dialysis, subjects onchemo-therapy and/or radiation therapy, transplant recipients, and thelike. Such individuals generally exhibit diminished immune responses tovaccines and therefore use of the compositions of the invention canenhance the immune responses achieved in these subjects.

In other embodiments, the antigen or antigens used in the compositionsof the invention include antigens associated with respiratory diseases,such as those caused or exacerbated by bacterial infection (e.g.pneumococcal), for the prophylaxis and therapy of conditions such aschronic obstructive pulmonary disease (COPD). COPD is definedphysiologically by the presence of irreversible or partially reversibleairway obstruction in patients with chronic bronchitis and/or emphysema(Am J Respir Crit. Care Med. 1995 November; 152(5 Pt 2):S77-121).Exacerbations of COPD are often caused by bacterial (e.g. pneumococcal)infection (Clin Microbiol Rev. 2001 April; 14(2):336-63). In aparticular embodiment, a composition of the invention comprises a GLAadjuvant, as described herein, in combination with the Pneumococcalvaccine Prevnar® (Wyeth).

In still other embodiments, the compositions of the invention,comprising GLA as described herein, are used in the treatment ofallergic conditions. For example, in a particular embodiment, thecompositions are used in allergy desensitization therapy. Such therapyinvolves the stimulation of the immune system with gradually increasingdoses of the substances to which a person is allergic, wherein thesubstances are formulated in compositions comprising GLA. In specificembodiments, the compositions are used in the treatment of allergies tofood products, pollen, mites, cats or stinging insects (e.g., bees,hornets, yellow jackets, wasps, velvet ants, fire ants).

TLR

As described herein, certain embodiments of the present inventioncontemplate vaccine compositions and immunological adjuvantcompositions, including pharmaceutical compositions, that include one ormore toll-like receptor agonist (TLR agonist). Toll-like receptors (TLR)include cell surface transmembrane receptors of the innate immune systemthat confer early-phase recognition capability to host cells for avariety of conserved microbial molecular structures such as may bepresent in or on a large number of infectious pathogens. (e.g., Armantet al., 2002 Genome Biol. 3(8):reviews 3011.1-3011.6; Fearon et al.,1996 Science 272:50; Medzhitov et al., 1997 Curr. Opin. Immunol. 9:4;Luster 2002 Curr. Opin. Immunol. 14:129; Lien et al. 2003 Nat. Immunol.4:1162; Medzhitov, 2001 Nat. Rev. Immunol. 1:135; Takeda et al., 2003Ann Rev Immunol. 21:335; Takeda et al. 2005 Int. Immunol. 17:1; Kaishoet al., 2004 Microbes Infect. 6:1388; Datta et al., 2003 J. Immunol.170:4102).

Induction of TLR-mediated signal transduction to potentiate theinitiation of immune responses via the innate immune system may beeffected by TLR agonists, which engage cell surface TLR. For example,lipopolysaccharide (LPS) may be a TLR agonist through TLR2 or TLR4 (Tsanet al., 2004 J. Leuk. Biol. 76:514; Tsan et al., 2004 Am. J. Physiol.Cell Phsiol. 286:C739; Lin et al., 2005 Shock 24:206);poly(inosine-cytidine) (polyl:C) may be a TLR agonist through TLR3(Salem et al., 2006 Vaccine 24:5119); CpG sequences(oligodeoxynucleotides containing unmethylated cytosine-guanosine or“CpG” dinucleotide motifs, e.g., CpG 7909, Cooper et al., 2005 AIDS19:1473; CpG 10101 Bayes et al. Methods Find Exp Clin Pharmacol 27:193;Vollmer et al. Expert Opinion on Biological Therapy 5:673; Vollmer etal., 2004 Antimicrob. Agents Chemother. 48:2314; Deng et al., 2004 J.Immunol. 173:5148) may be TLR agonists through TLR9 (Andaloussi et al.,2006 Glia 54:526; Chen et al., 2006 J. Immunol. 177:2373);peptidoglycans may be TLR2 and/or TLR6 agonists (Soboll et al., 2006Biol. Reprod. 75:131; Nakao et al., 2005 J. Immunol. 174:1566); 3M003(4-amino-2-(ethoxymethyl)-α,α-dimethyl-6,7,8,9-tetrahydro-1H-imidazo[4,5-c]quinoline-1-ethanolhydrate, Mol. Wt. 318 Da from 3M Pharmaceuticals, St. Paul, Minn., whichis also a source of the related compounds 3M001 and 3M002; Gorden etal., 2005 J. Immunol. 174:1259) may be a TLR7 agonist (Johansen 2005Clin. Exp. Allerg. 35:1591) and/or a TLR8 agonist (Johansen 2005);flagellin may be a TLR5 agonist (Feuillet et al., 2006 Proc. Nat. Acad.Sci. USA 103:12487); and hepatitis C antigens may act as TLR agoniststhrough TLR7 and/or TLR9 (Lee et al., 2006 Proc. Nat. Acad. Sci. USA103:1828; Horsmans et al., 2005 Hepatol. 42:724). Other TLR agonists areknown (e.g., Schirmbeck et al., 2003 J. Immunol. 171:5198) and may beused according to certain of the presently described embodiments.

For example, and by way of background (see, e.g., U.S. Pat. No.6,544,518) immunostimulatory oligonucleotides containing ummethylatedCpG dinucleotides (“CpG”) are known as being adjuvants when administeredby both systemic and mucosal routes (WO 96/02555, EP 468520, Davis etal., J. Immunol, 1998. 160(2):870-876; McCluskie and Davis, J. Immunol.,1998, 161(9):4463-6). CpG is an abbreviation for cytosine-guanosinedinucleotide motifs present in DNA. The central role of the CG motif inimmunostimulation was elucidated by Krieg, Nature 374, p 546 1995.Detailed analysis has shown that the CG motif has to be in a certainsequence context, and that such sequences are common in bacterial DNAbut are rare in vertebrate DNA. The immunostimulatory sequence is often:Purine, Purine, C, G, pyrimidine, pyrimidine; wherein the dinucleotideCG motif is not methylated, but other unmethylated CpG sequences areknown to be immunostimulatory and may be used in certain embodiments ofthe present invention. CpG when formulated into vaccines, may beadministered in free solution together with free antigen (WO 96/02555;McCluskie and Davis, supra) or covalently conjugated to an antigen (PCTPublication No. WO 98/16247), or formulated with a carrier such asaluminium hydroxide (e.g., Davis et al. supra, Brazolot-Millan et al.,Proc. Natl. Acad. Sci., USA, 1998, 95(26), 15553-8).

The preferred oligonucleotides for use in adjuvants or vaccines of thepresent invention preferably contain two or more dinucleotide CpG motifsseparated by at least three, more preferably at least six or morenucleotides. The oligonucleotides of the present invention are typicallydeoxynucleotides. In a preferred embodiment the internucleotide in theoligonucleotide is phosphorodithioate, or more preferably aphosphorothioate bond, although phosphodiester and other internucleotidebonds are within the scope of the invention including oligonucleotideswith mixed internucleotide linkages. Methods for producingphosphorothioate oligonucleotides or phosphorodithioate are described inU.S. Pat. Nos. 5,666,153, 5,278,302 and WO95/26204.

Examples of preferred oligonucleotides have sequences that are disclosedin the following publications; for certain herein disclosed embodimentsthe sequences preferably contain phosphorothioate modifiedinternucleotide linkages:

CPG 7909: Cooper et al., “CPG 7909 adjuvant improves hepatitis B virusvaccine seroprotection in antiretroviral-treated HIV-infected adults.”AIDS, 2005 Sep. 23; 19(14):1473-9.

CpG 10101: Bayes et al., “Gateways to clinical trials.” Methods Find.Exp. Clin. Pharmacol. 2005 April; 27(3):193-219.

Vollmer J., “Progress in drug development of immunostimula-tory CpGoligodeoxynucleotide ligands for TLR9.” Expert Opinion on BiologicalTherapy. 2005 May; 5(5): 673-682

Alternative CpG oligonucleotides may comprise variants of the preferredsequences described in the above-cited publications that differ in thatthey have inconsequential nucleotide sequence substitutions, insertions,deletions and/or additions thereto. The CpG oligonucleotides utilized incertain embodiments of the present invention may be synthesized by anymethod known in the art (e.g., EP 468520). Conveniently, sucholigonucleotides may be synthesized utilising an automated synthesizer.The oligonucleotides are typically deoxynucleotides. In a preferredembodiment the internucleotide bond in the oligonucleotide isphosphorodithioate, or more preferably phosphorothioate bond, althoughphosphodiesters are also within the scope of the presently contemplatedembodiments. Oligonucleotides comprising different internucleotidelinkages are also contemplated, e.g., mixed phosphorothioatephophodiesters. Other internucleotide bonds which stabilize theoligonucleotide may also be used.

Co-Adjuvant

Certain embodiments as provided herein include vaccine compositions andimmunological adjuvant compositions, including pharmaceuticalcompositions, that contain, in addition to GLA, at least oneco-adjuvant, which refers to a component of such compositions that hasadjuvant activity but that is other than GLA. A co-adjuvant having suchadjuvant activity includes a composition that, when administered to asubject such as a human (e.g., a human patient), a non-human primate, amammal or another higher eukaryotic organism having a recognized immunesystem, is capable of altering (i.e., increasing or decreasing in astatistically significant manner, and in certain preferred embodiments,enhancing or increasing) the potency and/or longevity of an immuneresponse. (See, e.g., Powell and Newman, “Vaccine design—The Subunit andAdjuvant Approach”, 1995, Plenum Press, New York) In certain embodimentsdisclosed herein GLA and a desired antigen, and optionally one or moreco-adjuvants, may so alter, e.g., elicit or enhance, an immune responsethat is directed against the desired antigen which may be administeredat the same time as GLA or may be separated in time and/or space (e.g.,at a different anatomic site) in its administration, but certaininvention embodiments are not intended to be so limited and thus alsocontemplate administration of GLA in a composition that does not includea specified antigen but which may also include one or more of a TLRagonist, a co-adjuvant, an imidazoquinline immune response modifier, anda double stem loop immune modifier (dSLIM).

Accordingly and as noted above, co-adjuvants include compositions otherthan GLA that have adjuvant effects, such as saponins and saponinmimetics, including QS21 and QS21 mimetics (see, e.g., U.S. Pat. No.5,057,540; EP 0 362 279 B1; WO 95/17210), alum, plant alkaloids such astomatine, detergents such as (but not limited to) saponin, polysorbate80, Span 85 and stearyl tyrosine, one or more cytokines (e.g., GM-CSF,IL-2, IL-7, IL-12, TNF-alpha, IFN-gamma), an imidazoquinoline immuneresponse modifier, and a double stem loop immune modifier (dSLIM, e.g.,Weeratna et al., 2005 Vaccine 23:5263).

Detergents including saponins are taught in, e.g., U.S. Pat. No.6,544,518; Lacaille-Dubois, M and Wagner H. (1996 Phytomedicine2:363-386), U.S. Pat. No. 5,057,540, Kensil, Crit. Rev Ther Drug CarrierSyst, 1996, 12 (1-2):1-55, and EP 0 362 279 B1. Particulate structures,termed Immune Stimulating Complexes (ISCOMS), comprising fractions ofQuil A (saponin) are haemolytic and have been used in the manufacture ofvaccines (Morein, B., EP 0 109 942 B1). These structures have beenreported to have adjuvant activity (EP 0 109 942 B1; WO 96/11711). Thehaemolytic saponins QS21 and QS17 (HPLC purified fractions of Quil A)have been described as potent systemic adjuvants, and the method oftheir production is disclosed in U.S. Pat. No. 5,057,540 and EP 0 362279 B1. Also described in these references is the use of QS7 (anon-haemolytic fraction of Quil-A) which acts as a potent adjuvant forsystemic vaccines. Use of QS21 is further described in Kensil et al.(1991. J. Immunology 146:431-437). Combinations of QS21 and polysorbateor cyclodextrin are also known (WO 99/10008). Particulate adjuvantsystems comprising fractions of QuilA, such as QS21 and QS7 aredescribed in WO 96/33739 and WO 96/11711. Other saponins which have beenused in systemic vaccination studies include those derived from otherplant species such as Gypsophila and Saponaria (Bomford et al., Vaccine,10(9):572-577, 1992).

Escin is another detergent related to the saponins for use in theadjuvant compositions of the embodiments herein disclosed. Escin isdescribed in the Merck index (12^(th) Ed.: entry 3737) as a mixture ofsaponin occurring in the seed of the horse chestnut tree, Aesculushippocastanum. Its isolation is described by chromatography andpurification (Fiedler, Arzneimittel-Forsch. 4, 213 (1953)), and byion-exchange resins (Erbring et al., U.S. Pat. No. 3,238,190). Fractionsof escin (also known as aescin) have been purified and shown to bebiologically active (Yoshikawa M, et al. (Chem Pharm Bull (Tokyo) 1996August; 44(8): 1454-1464)). Digitonin is another detergent, also beingdescribed in the Merck index (12th Ed., entry 3204) as a saponin, beingderived from the seeds of Digitalis purpurea and purified according tothe procedure described by Gisvold et al., J. Am. Pharm. Assoc., 1934,23, 664; and Rubenstroth-Bauer, Physiol. Chem., 1955, 301, 621.

Other co-adjuvants for use according to certain herein disclosedembodiments include a block co-polymer or biodegradable polymer, whichrefers to a class of polymeric compounds with which those in therelevant art will be familiar. Examples of a block co-polymer orbiodegradable polymer that may be included in a GLA vaccine compositionor a GLA immunological adjuvant include Pluronic® L121 (BASF Corp.,Mount Olive, N.J.; see, e.g., Yeh et al., 1996 Pharm. Res. 13:1693; U.S.Pat. No. 5,565,209), CRL1005 (e.g., Triozzi et al., 1997 Clin Canc. Res.3:2355), poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA),poly-(D,L-lactide-co-glycolide) (PLG), and polyl:C. (See, e.g., Powelland Newman, “Vaccine design—The Subunit and Adjuvant Approach”, 1995,Plenum Press, New York)

Certain embodiments contemplate GLA vaccines and GLA immunologicaladjuvants that include an oil, which in some such embodiments maycontribute co-adjuvant activity and in other such embodiments mayadditionally or alternatively provide a pharmaceutically acceptablecarrier or excipient. Any number of suitable oils are known and may beselected for inclusion in vaccine compositions and immunologicaladjuvant compositions based on the present disclosure. Examples of suchoils, by way of illustration and not limitation, include squalene,squalane, mineral oil, olive oil, cholesterol, and a mannide monooleate.

Immune response modifiers such as imidazoquinoline immune responsemodifiers are also known in the art and may also be included asco-adjuvants in certain presently disclosed embodiments. Certainpreferred imidazoquinoline immune response modifiers include, by way ofnon-limiting example, resiquimod (R848), imiquimod and gardiquimod(Hemmi et al., 2002 Nat. Immunol. 3:196; Gibson et al., 2002 Cell.Immunol. 218:74; Gorden et al., 2005 J. Immunol. 174:1259); these andother imidazoquinoline immune response modifiers may, under appropriateconditions, also have TLR agonist activity as described herein. Otherimmune response modifiers are the nucleic acid-based double stem loopimmune modifiers (dSLIM). Specific examples of dSLIM that arecontemplated for use in certain of the presently disclosed embodimentscan be found in Schmidt et al., 2006 Allergy 61:56; Weihrauch et al.2005 Clin Cancer Res. 11(16):5993-6001; Modern Biopharmaceuticals, J.Knäblein (Editor). John Wiley & Sons, Dec. 6, 2005. (dSLIM discussed onpages 183 to ˜200), and from Mologen AG (Berlin, FRG: [retrieved onlineon Aug. 18, 2006 at http://www.mologen.com/English/04.20-dSLIM.shtml].

As also noted above, one type of co-adjuvant for use with GLA asdescribed herein may be the aluminum co-adjuvants, which are generallyreferred to as “alum.” Alum co-adjuvants are based on the following:aluminum oxy-hydroxide; aluminum hydroxyphosphoate; or variousproprietary salts. Vaccines that use alum co-adjuvants may includevaccines for tetanus strains, HPV, hepatitis A, inactivated polio virus,and other antigens as described herein. Alum co-adjuvants areadvantageous because they have a good safety record, augment antibodyresponses, stabilize antigens, and are relatively simple for large-scaleproduction. (Edelman 2002 Mol. Biotechnol. 21:129-148; Edelman, R. 1980Rev. Infect. Dis. 2:370-383.)

Other co-adjuvants that may be combined with GLA for effective immunestimulation include saponins and saponin mimetics, including QS21 andstructurally related compounds conferring similar effects and referredto herein as QS21 mimetics. QS21 has been recognized as a preferredco-adjuvant. QS21 may comprise an HPLC purified non-toxic fractionderived from the bark of Quillaja Saponaria Molina. The production ofQS21 is disclosed in U.S. Pat. No. 5,057,540. (See also U.S. Pat. Nos.6,936,255, 7,029,678 and 6,932,972.)

GLA may also in certain embodiments be combined with “immunostimulatorycomplexes” known as ISCOMS (e.g., U.S. Pat. Nos. 6,869,607, 6,846,489,6,027,732, 4,981,684), including saponin-derived ISCOMATRIX®, which iscommercially available, for example, from Iscotec (Stockholm, Sweden)and CSL Ltd. (Parkville, Victoria, Australia).

In still other embodiments, adjuvant/delivery systems may be used incombination with GLA which are lipid assemblies (liposomes and otherformulations) comprising, for example, positively charged polycationiclipids. Illustratively, such systems may include a syntheticbiocompatible polycationic sphingolipid (e.g., D-Erythro-N-palmitoylsphingosyl-1-0 carbamoyl-spermine; also referred to as CCS; availablefrom NasVax Ltd.) or may include cholesterol (C) in addition to the CCSlipid (referred to as CCS/C or Vaxisome®; available from NasVax Ltd.).The vaccine liposomal formulations may be formed, for example, by mixinga dry powder of CCS or CCS/C with vaccine proteins or polynucleotides(antigens), such as in aqueous suspension. Antigen-loaded liposomes canthen be sprayed into the nose (i.n.) or injected intramuscularly (i.m.)or subcutaneously (s.c.), for example.

In further embodiments, a vesicular adjuvant/delivery system consistingof unilamellar or multilamellar vesicles, called niosomes, can be usedin conjunction with GLA. In this case, an aqueous solution is generallyenclosed in a highly ordered bilayer made up of non-ionic surfactant,with or without cholesterol and dicetyl phosphate, and exhibit abehaviour similar to liposomes in vivo. The bilayered vesicularstructure is an assembly of hydrophobic tails of surfactant monomer,shielded away from the aqueous space located in the center andhydrophilic head group, in contact with the same. Addition ofcholesterol results in an ordered liquid phase formation which gives therigidity to the bilayer, and results in less leaky niosomes. Dicetylphosphate is known to increase the size of vesicles, provide charge tothe vesicles, and thus shows increase entrapment efficiency. Othercharge-inducers are stearylamine and diacylglycerol, that also help inelectrostatic stabilization of the vesicles.

Niosomes have unique advantages over liposomes. Nisomes are quite stablestructures, even in the emulsified form. They require no specialconditions such as low temperature or inert atmosphere for protection orstorage, and are chemically stable. Relatively low cost of materialsmakes it suitable for industrial manufacture. A number of non-ionicsurfactants have been used to prepare vesicles, e.g., polyglycerol alkylether, glucosyl dialkyl ethers, crown ethers, ester linked surfactants,polyoxyethylene alkyl ether, Brij, and various spans and tweens.

Similar to liposomes, there are 3 major types of niosomes—multilamellarvesicles (MLV, size >0.05 μm), small unilamellar vesicles (SUV, size−0.025-0.05 μm), and large unilamellar vesicles (LUV, size >0.10 μm).MLVs vesicles exhibit increased-trapped volume and equilibrium solutedistribution, and generally require hand-shaking method. They showvariations in lipid compositions. SUVs are commonly produced bysonication, and French Press procedures. Ultrasonic electrocapillaryemulsification or solvent dilution techniques can also be used toprepare SUVs. The injections of lipids solubilized in an organic solventinto an aqueous buffer can result in spontaneous formation of LUV.Another method of preparation of LUV is reverse phase evaporation, or bydetergent solubilization method.

Recombinant Expression Construct

According to certain herein disclosed embodiments, the GLA vaccinecomposition may contain at least one recombinant expression constructwhich comprises a promoter operably linked to a nucleic acid sequenceencoding an antigen. In certain further embodiments the recombinantexpression construct is present in a viral vector, such as anadenovirus, adeno-associated virus, herpesvirus, lentivirus, poxvirus orretrovirus vector. Compositions and methods for making and using suchexpression constructs and vectors are known in the art, for theexpression of polypeptide antigens as provided herein, for example,according to Ausubel et al. (Eds.), Current Protocols in MolecularBiology, 2006 John Wiley & Sons, NY. Non-limiting examples ofrecombinant expression constructs generally can be found, for instance,in U.S. Pat. Nos. 6,844,192; 7,037,712; 7,052,904; 7,001,770; 6,106,824;5,693,531; 6,613,892; 6,875,610; 7,067,310; 6,218,186; 6,783,981;7,052,904; 6,783,981; 6,734,172; 6,713,068; 5,795,577 and 6,770,445 andelsewhere, with teachings that can be adapted to the expression ofpolypeptide antigens as provided herein, for use in certain presentlydisclosed embodiments.

Immune Response

The invention thus provides compositions for altering (i.e., increasingor decreasing in a statistically significant manner, for example,relative to an appropriate control as will be familiar to personsskilled in the art) immune responses in a host capable of mounting animmune response. As will be known to persons having ordinary skill inthe art, an immune response may be any active alteration of the immunestatus of a host, which may include any alteration in the structure orfunction of one or more tissues, organs, cells or molecules thatparticipate in maintenance and/or regulation of host immune status.Typically, immune responses may be detected by any of a variety of wellknown parameters, including but not limited to in vivo or in vitrodetermination of: soluble immunoglobulins or antibodies; solublemediators such as cytokines, lymphokines, chemokines, hormones, growthfactors and the like as well as other soluble small peptide,carbohydrate, nucleotide and/or lipid mediators; cellular activationstate changes as determined by altered functional or structuralproperties of cells of the immune system, for example cellproliferation, altered motility, induction of specialized activitiessuch as specific gene expression or cytolytic behavior; cellulardifferentiation by cells of the immune system, including altered surfaceantigen expression profiles or the onset of apoptosis (programmed celldeath); or any other criterion by which the presence of an immuneresponse may be detected.

Immune responses may often be regarded, for instance, as discriminationbetween self and non-self structures by the cells and tissues of ahost's immune system at the molecular and cellular levels, but theinvention should not be so limited. For example, immune responses mayalso include immune system state changes that result from immunerecognition of self molecules, cells or tissues, as may accompany anynumber of normal conditions such as typical regulation of immune systemcomponents, or as may be present in pathological conditions such as theinappropriate autoimmune responses observed in autoimmune anddegenerative diseases. As another example, in addition to induction byup-regulation of particular immune system activities (such as antibodyand/or cytokine production, or activation of cell mediated immunity)immune responses may also include suppression, attenuation or any otherdown-regulation of detectable immunity, which may be the consequence ofthe antigen selected, the route of antigen administration, specifictolerance induction or other factors.

Determination of the induction of an immune response by the vaccines ofthe present invention may be established by any of a number of wellknown immunological assays with which those having ordinary skill in theart will be readily familiar. Such assays include, but need not belimited to, to in vivo or in vitro determination of: soluble antibodies;soluble mediators such as cytokines, lymphokines, chemokines, hormones,growth factors and the like as well as other soluble small peptide,carbohydrate, nucleotide and/or lipid mediators; cellular activationstate changes as determined by altered functional or structuralproperties of cells of the immune system, for example cellproliferation, altered motility, induction of specialized activitiessuch as specific gene expression or cytolytic behavior; cellulardifferentiation by cells of the immune system, including altered surfaceantigen expression profiles or the onset of apoptosis (programmed celldeath). Procedures for performing these and similar assays are widelyknown and may be found, for example in Lefkovits (Immunology MethodsManual: The Comprehensive Sourcebook of Techniques, 1998; see alsoCurrent Protocols in Immunology; see also, e.g., Weir, Handbook ofExperimental Immunology, 1986 Blackwell Scientific, Boston, Mass.;Mishell and Shigii (eds.) Selected Methods in Cellular Immunology, 1979Freeman Publishing, San Francisco, Calif.; Green and Reed, 1998 Science281:1309 and references cited therein.).

Detection of the proliferation of antigen-reactive T cells may beaccomplished by a variety of known techniques. For example, T cellproliferation can be detected by measuring the rate of DNA synthesis,and antigen specificity can be determined by controlling the stimuli(such as, for example, a specific desired antigen- or a controlantigen-pulsed antigen presenting cells) to which candidateantigen-reactive T cells are exposed. T cells which have been stimulatedto proliferate exhibit an increased rate of DNA synthesis. A typical wayto measure the rate of DNA synthesis is, for example, by pulse-labelingcultures of T cells with tritiated thymidine, a nucleoside precursorwhich is incorporated into newly synthesized DNA. The amount oftritiated thymidine incorporated can be determined using a liquidscintillation spectrophotometer. Other ways to detect T cellproliferation include measuring increases in interleukin-2 (IL-2)production, Ca²⁺ flux, or dye uptake, such as3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium. Alternatively,synthesis of lymphokines (such as interferon-gamma) can be measured orthe relative number of T cells that can respond to a particular antigenmay be quantified.

Detection of antigen-specific antibody production may be achieved, forexample, by assaying a sample (e.g., an immunoglobulin containing samplesuch as serum, plasma or blood) from a host treated with a vaccineaccording to the present invention using in vitro methodologies such asradioimmunoassay (RIA), enzyme linked immunosorbent assays (ELISA),equilibrium dialysis or solid phase immunoblotting including Westernblotting. In preferred embodiments ELISA assays may further includeantigen-capture immobilization of the target antigen with a solid phasemonoclonal antibody specific for the antigen, for example, to enhancethe sensitivity of the assay. Elaboration of soluble mediators (e.g.,cytokines, chemokines, lymphokines, prostaglandins, etc.) may also bereadily determined by enzyme-linked immunosorbent assay (ELISA), forexample, using methods, apparatus and reagents that are readilyavailable from commercial sources (e.g., Sigma, St. Louis, Mo.; see alsoR & D Systems 2006 Catalog, R & D Systems, Minneapolis, Minn.).

Any number of other immunological parameters may be monitored usingroutine assays that are well known in the art. These may include, forexample, antibody dependent cell-mediated cytotoxicity (ADCC) assays,secondary in vitro antibody responses, flow immunocytofluorimetricanalysis of various peripheral blood or lymphoid mononuclear cellsubpopulations using well established marker antigen systems,immunohistochemistry or other relevant assays. These and other assaysmay be found, for example, in Rose et al. (Eds.), Manual of ClinicalLaboratory Immunology, 5^(th) Ed., 1997 American Society ofMicrobiology, Washington, D.C.

Accordingly it is contemplated that the vaccine and adjuvantcompositions provided herein will be capable of eliciting or enhancingin a host at least one immune response that is selected from aT_(H)1-type T lymphocyte response, a T_(H)2-type T lymphocyte response,a cytotoxic T lymphocyte (CTL) response, an antibody response, acytokine response, a lymphokine response, a chemokine response, and aninflammatory response. In certain embodiments the immune response maycomprise at least one of production of one or a plurality of cytokineswherein the cytokine is selected from interferon-gamma (IFN-γ), tumornecrosis factor-alpha (TNF-α), production of one or a plurality ofinterleukins wherein the interleukin is selected from IL-1, IL-2, IL-3,IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-16, IL-18 and IL-23,production one or a plurality of chemokines wherein the chemokine isselected from MIP-1α, MIP-1β, RANTES, CCL4 and CCL5, and a lymphocyteresponse that is selected from a memory T cell response, a memory B cellresponse, an effector T cell response, a cytotoxic T cell response andan effector B cell response. See, e.g., WO 94/00153; WO 95/17209; WO96/02555; U.S. Pat. No. 6,692,752; U.S. Pat. No. 7,084,256; U.S. Pat.No. 6,977,073; U.S. Pat. No. 6,749,856; U.S. Pat. No. 6,733,763; U.S.Pat. No. 6,797,276; U.S. Pat. No. 6,752,995; U.S. Pat. No. 6,057,427;U.S. Pat. No. 6,472,515; U.S. Pat. No. 6,309,847; U.S. Pat. No.6,969,704; U.S. Pat. No. 6,120,769; U.S. Pat. No. 5,993,800; U.S. Pat.No. 5,595,888; Smith et al., 1987 J Biol Chem. 262:6951; Kriegler etal., 1988 Cell 53:45 53;Beutler et al., 1986 Nature 320:584; U.S. Pat.No. 6,991,791; U.S. Pat. No. 6,654,462; U.S. Pat. No. 6,375,944.

Pharmaceutical Compositions

Pharmaceutical compositions generally comprise GLA (available fromAvanti Polar Lipids, Inc., Alabaster, Ala.; product number 699800) andmay further comprise one or more components as provided herein that areselected from antigen, TLR agonist, co-adjuvant (including optionally acytokine, an imidazoquinoline immune response modifier and/or a dSLIM),and/or a recombinant expression construct, in combination with apharmaceutically acceptable carrier, excipient or diluent.

Therefore, in certain aspects, the present invention is drawn to GLA“monotherapy” wherein GLA, as described herein, is formulated in acomposition that is substantially devoid of other antigens, and isadministered to a subject in order to stimulate an immune response,e.g., a non-specific immune response, for the purpose of treating orpreventing a disease or other condition, such as for treating aninfection by an organism, for treating seasonal rhinitis, or the like.In one embodiment, for example, the compositions and methods of theinvention employ a monophosphorylated disaccharide for stimulating animmune response in a subject. In another particular embodiment, thecompositions and methods employ a 2-monoacyl form of Lipid A forstimulating an immune response in a subject. In another particularembodiment, the GLA is in the form of a spray, optionally provided in akit.

The GLA may be preferably formulated in a stable emulsion. In oneparticular embodiment, for example, a composition is provided comprisinga lipid A derivative in a stable emulsion substantially devoid of otherantigens. In another particular embodiment, a composition is providedcomprising a derivative of 3-acylated monophosphorylated lipid A,suitable for use in mammals, wherein the 2 amine position has a singleacyl chain, and that is substantially devoid of other antigens.

In certain other embodiments, the pharmaceutical composition is avaccine composition that comprises both GLA and an antigen and mayfurther comprise one or more components, as provided herein, that areselected from TLR agonist, co-adjuvant (including, e.g., a cytokine, animidazoquinoline immune response modifier and/or a dSLIM) and the likeand/or a recombinant expression construct, in combination with apharmaceutically acceptable carrier, excipient or diluent.

Illustrative carriers will be nontoxic to recipients at the dosages andconcentrations employed. For GLA-plus-nucleic acid-based vaccines, orfor vaccines comprising GLA plus an antigen, about 0.01 μg/kg to about100 mg/kg body weight will be administered, typically by theintradermal, subcutaneous, intramuscular or intravenous route, or byother routes.

A preferred dosage is about 1 μg/kg to about 1 mg/kg, with about 5 μg/kgto about 200 μg/kg particularly preferred. It will be evident to thoseskilled in the art that the number and frequency of administration willbe dependent upon the response of the host. “Pharmaceutically acceptablecarriers” for therapeutic use are well known in the pharmaceutical art,and are described, for example, in Remingtons Pharmaceutical Sciences,Mack Publishing Co. (A. R. Gennaro edit. 1985). For example, sterilesaline and phosphate-buffered saline at physiological pH may be used.Preservatives, stabilizers, dyes and even flavoring agents may beprovided in the pharmaceutical composition. For example, sodiumbenzoate, sorbic acid and esters of p-hydroxybenzoic acid may be addedas preservatives. Id. at 1449. In addition, antioxidants and suspendingagents may be used. Id.

“Pharmaceutically acceptable salt” refers to salts of the compounds ofthe present invention derived from the combination of such compounds andan organic or inorganic acid (acid addition salts) or an organic orinorganic base (base addition salts). The compositions of the presentinvention may be used in either the free base or salt forms, with bothforms being considered as being within the scope of the presentinvention.

The pharmaceutical compositions may be in any form which allows for thecomposition to be administered to a patient. For example, thecomposition may be in the form of a solid, liquid or gas (aerosol).Typical routes of administration include, without limitation, oral,topical, parenteral (e.g., sublingually or buccally), sublingual,rectal, vaginal, and intranasal (e.g., as a spray). The term parenteralas used herein includes iontophoretic (e.g., U.S. Pat. Nos. 7,033,598;7,018,345; 6,970,739), sonophoretic (e.g., U.S. Pat. Nos. 4,780,212;4,767,402; 4,948,587; 5,618,275; 5,656,016; 5,722,397; 6,322,532;6,018,678), thermal (e.g., U.S. Pat. Nos. 5,885,211; 6,685,699), passivetransdermal (e.g., U.S. Pat. Nos. 3,598,122; 3,598,123; 4,286,592;4,314,557; 4,379,454; 4,568,343; 5,464,387; UK Pat. Spec. No. 2232892;U.S. Pat. Nos. 6,871,477; 6,974,588; 6,676,961), microneedle (e.g., U.S.Pat. Nos. 6,908,453; 5,457,041; 5,591,139; 6,033,928) administration andalso subcutaneous injections, intravenous, intramuscular, intrasternal,intracavernous, intrathecal, intrameatal, intraurethral injection orinfusion techniques. In a particular embodiment, a composition asdescribed herein (including vaccine and pharmaceutical compositions) isadministered intradermally by a technique selected from iontophoresis,microcavitation, sonophoresis or microneedles.

The pharmaceutical composition is formulated so as to allow the activeingredients contained therein to be bioavailable upon administration ofthe composition to a patient. Compositions that will be administered toa patient take the form of one or more dosage units, where for example,a tablet may be a single dosage unit, and a container of one or morecompounds of the invention in aerosol form may hold a plurality ofdosage units.

For oral administration, an excipient and/or binder may be present.Examples are sucrose, kaolin, glycerin, starch dextrins, sodiumalginate, carboxymethylcellulose and ethyl cellulose. Coloring and/orflavoring agents may be present. A coating shell may be employed.

The composition may be in the form of a liquid, e.g., an elixir, syrup,solution, emulsion or suspension. The liquid may be for oraladministration or for delivery by injection, as two examples. Whenintended for oral administration, preferred compositions contain one ormore of a sweetening agent, preservatives, dye/colorant and flavorenhancer. In a composition intended to be administered by injection, oneor more of a surfactant, preservative, wetting agent, dispersing agent,suspending agent, buffer, stabilizer and isotonic agent may be included.

A liquid pharmaceutical composition as used herein, whether in the formof a solution, suspension or other like form, may include one or more ofthe following carriers or excipients: sterile diluents such as water forinjection, saline solution, preferably physiological saline, Ringer'ssolution, isotonic sodium chloride, fixed oils such as squalene,squalane, mineral oil, a mannide monooleate, cholesterol, and/orsynthetic mono or digylcerides which may serve as the solvent orsuspending medium, polyethylene glycols, glycerin, propylene glycol orother solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic. An injectable pharmaceutical composition ispreferably sterile.

In a particular embodiment, a pharmaceutical or vaccine composition ofthe invention comprises a stable aqueous suspension of less than 0.2 umand further comprises at least one component selected from the groupconsisting of phospholipids, fatty acids, surfactants, detergents,saponins, fluorodated lipids, and the like.

In another embodiment, a composition of the invention is formulated in amanner which can be aerosolized.

It may also be desirable to include other components in a vaccine orpharmaceutical composition, such as delivery vehicles including but notlimited to aluminum salts, water-in-oil emulsions, biodegradable oilvehicles, oil-in-water emulsions, biodegradable microcapsules, andliposomes. Examples of additional immunostimulatory substances(co-adjuvants) for use in such vehicles are also described above and mayinclude N-acetylmuramyl-L-alanine-D-isoglutamine (MDP), glucan, IL-12,GM-CSF, gamma interferon and IL-12.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will vary depending on the mode of administrationand whether a sustained release is desired. For parenteraladministration, such as subcutaneous injection, the carrier preferablycomprises water, saline, alcohol, a fat, a wax or a buffer. For oraladministration, any of the above carriers or a solid carrier, such asmannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, sucrose, and magnesium carbonate, may beemployed. Biodegradable microspheres (e.g., polylactic galactide) mayalso be employed as carriers for the pharmaceutical compositions of thisinvention. Suitable biodegradable microspheres are disclosed, forexample, in U.S. Pat. Nos. 4,897,268 and 5,075,109. In this regard, itis preferable that the microsphere be larger than approximately 25microns.

Pharmaceutical compositions (including GLA vaccines and GLAimmunological adjuvants) may also contain diluents such as buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, proteins, amino acids, carbohydratesincluding glucose, sucrose or dextrins, chelating agents such as EDTA,glutathione and other stabilizers and excipients. Neutral bufferedsaline or saline mixed with nonspecific serum albumin are exemplaryappropriate diluents. Preferably, product may be formulated as alyophilizate using appropriate excipient solutions (e.g., sucrose) asdiluents.

As described above, in certain embodiments the subject inventionincludes compositions capable of delivering nucleic acid moleculesencoding desired antigens. Such compositions include recombinant viralvectors (e.g., retroviruses (see WO 90/07936, WO 91/02805, WO 93/25234,WO 93/25698, and

WO 94/03622), adenovirus (see Berkner, Biotechniques 6:616-627, 1988; Liet al., Hum. Gene Ther. 4:403-409, 1993; Vincent et al., Nat. Genet.5:130-134, 1993; and Kolls et al., Proc. Natl. Acad. Sci. USA91:215-219, 1994), pox virus (see U.S. Pat. No. 4,769,330; U.S. Pat. No.5,017,487; and WO 89/01973)), recombinant expression construct nucleicacid molecules complexed to a polycationic molecule (see WO 93/03709),and nucleic acids associated with liposomes (see Wang et al., Proc.Natl. Acad. Sci. USA 84:7851, 1987). In certain embodiments, the DNA maybe linked to killed or inactivated adenovirus (see Curiel et al., Hum.Gene Ther. 3:147-154, 1992; Cotton et al., Proc. Natl. Acad. Sci. USA89:6094, 1992). Other suitable compositions include DNA-ligand (see Wuet al., J. Biol. Chem. 264:16985-16987, 1989) and lipid-DNA combinations(see Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1989).

In addition to direct in vivo procedures, ex vivo procedures may be usedin which cells are removed from a host, modified, and placed into thesame or another host animal. It will be evident that one can utilize anyof the compositions noted above for introduction of antigen-encodingnucleic acid molecules into tissue cells in an ex vivo context.Protocols for viral, physical and chemical methods of uptake are wellknown in the art.

Accordingly, the present invention is useful for enhancing or eliciting,in a host, a patient or in cell culture, an immune response. As usedherein, the term “patient” refers to any warm-blooded animal, preferablya human. A patient may be afflicted with an infectious disease, cancer,such as breast cancer, or an autoimmune disease, or may be normal (i.e.,free of detectable disease and/or infection). A “cell culture” is anypreparation containing immunocompetent cells or isolated cells of theimmune system (including, but not limited to, T cells, macrophages,monocytes, B cells and dendritic cells). Such cells may be isolated byany of a variety of techniques well known to those of ordinary skill inthe art (e.g., Ficoll-hypaque density centrifugation). The cells may(but need not) have been isolated from a patient afflicted with cancer,and may be reintroduced into a patient after treatment.

In certain embodiments a liquid composition intended for eitherparenteral or oral administration should contain an amount of GLAvaccine composition such that a suitable dosage will be obtained.Typically, this amount is at least 0.01 wt % of an antigen in thecomposition. When intended for oral administration, this amount may bevaried to be between 0.1 and about 70% of the weight of the composition.Preferred oral compositions contain between about 4% and about 50% ofthe antigen. Preferred compositions and preparations are prepared sothat a parenteral dosage unit contains between 0.01 to 1% by weight ofactive composition.

The pharmaceutical composition may be intended for topicaladministration, in which case the carrier may suitably comprise asolution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, beeswax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device. Topical formulations may contain aconcentration of the antigen (e.g., GLA-antigen vaccine composition) orGLA (e.g., immunological adjuvant composition; GLA is available fromAvanti Polar Lipids, Inc., Alabaster, Ala.; e.g., product number 699800)of from about 0.1 to about 10% w/v (weight per unit volume).

The composition may be intended for rectal administration, in the form,e.g., of a suppository which will melt in the rectum and release thedrug. The composition for rectal administration may contain anoleaginous base as a suitable nonirritating excipient. Such basesinclude, without limitation, lanolin, cocoa butter and polyethyleneglycol. In the methods of the invention, the vaccinecompositions/adjuvants may be administered through use of insert(s),bead(s), timed-release formulation(s), patch(es) or fast-releaseformulation(s).

Also contemplated in certain embodiments are kits comprising the hereindescribed GLA vaccine compositions and/or GLA immunological adjuvantcompositions, which may be provided in one or more containers. In oneembodiment all components of the GLA vaccine compositions and/or GLAimmunological adjuvant compositions are present together in a singlecontainer, but the invention embodiments are not intended to be solimited and also contemplate two or more containers in which, forexample, a GLA immunological adjuvant composition is separate from, andnot in contact with, the antigen component. By way of non-limitingtheory, it is believed that in some cases administration only of the GLAimmunological adjuvant composition may be performed beneficially, whilstin other cases such administration may beneficially be separatedtemporally and/or spatially (e.g., at a different anatomical site) fromadministration of the antigen, whilst in still other casesadministration to the subject is beneficially conducted of a GLA vaccinecomposition as described herein and containing both antigen and GLA, andoptionally other herein described components as well.

A container according to such kit embodiments may be any suitablecontainer, vessel, vial, ampule, tube, cup, box, bottle, flask, jar,dish, well of a single-well or multi-well apparatus, reservoir, tank, orthe like, or other device in which the herein disclosed compositions maybe placed, stored and/or transported, and accessed to remove thecontents. Typically such a container may be made of a material that iscompatible with the intended use and from which recovery of thecontained contents can be readily achieved. Preferred examples of suchcontainers include glass and/or plastic sealed or re-sealable tubes andampules, including those having a rubber septum or other sealing meansthat is compatible with withdrawal of the contents using a needle andsyringe. Such containers may, for instance, by made of glass or achemically compatible plastic or resin, which may be made of, or may becoated with, a material that permits efficient recovery of material fromthe container and/or protects the material from, e.g., degradativeconditions such as ultraviolet light or temperature extremes, or fromthe introduction of unwanted contaminants including microbialcontaminants. The containers are preferably sterile or sterilizable, andmade of materials that will be compatible with any carrier, excipient,solvent, vehicle or the like, such as may be used to suspend or dissolvethe herein described vaccine compositions and/or immunological adjuvantcompositions and/or antigens and/or recombinant expression constructs,etc.

Emulsion systems may also be used in formulating compositions of thepresent invention. For example, many single or multiphase emulsionsystems have been described. Oil in water emulsion adjuvants per se havebeen suggested to be useful as adjuvant composition (EP 0 399 843B),also combinations of oil in water emulsions and other active agents havebeen described as adjuvants for vaccines (WO 95/17210; WO 98/56414; WO99/12565; WO 99/11241). Other oil emulsion adjuvants have beendescribed, such as water in oil emulsions (U.S. Pat. No. 5,422,109; EP 0480 982 B2) and water in oil in water emulsions (U.S. Pat. No.5,424,067; EP 0 480 981 B). The oil emulsion adjuvants for use in thepresent invention may be natural or synthetic, and may be mineral ororganic. Examples of mineral and organic oils will be readily apparentto the man skilled in the art.

In a particular embodiment, a composition of the invention comprises anemulsion of oil in water wherein the GLA is incorporated in the oilphase. In another embodiment, a composition of the invention comprisesan emulsion of oil in water wherein the GLA is incorporated in the oilphase and wherein an additional component is present, such as aco-adjuvant, TLR agonist, or the like, as described herein.

In order for any oil in water composition to be suitable for humanadministration, the oil phase of the emulsion system preferablycomprises a metabolizable oil. The meaning of the term metabolizable oilis well known in the art. Metabolizable can be defined as “being capableof being transformed by metabolism” (Dorland's illustrated MedicalDictionary, W. B. Saunders Company, 25th edition (1974)). The oil may beany vegetable oil, fish oil, animal oil or synthetic oil, which is nottoxic to the recipient and is capable of being transformed bymetabolism. Nuts (such as peanut oil), seeds, and grains are commonsources of vegetable oils. Synthetic oils are also part of thisinvention and can include commercially available oils such as NEOBEE®and others.

Squalene (2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22-tetracosahexaene),for example, is an unsaturated oil which is found in large quantities inshark-liver oil, and in lower quantities in olive oil, wheat germ nil,rice bran oil, and yeast, and is a particularly preferred oil for use inthis invention. Squalene is a metabolizable oil virtue of the fact thatit is an intermediate in the biosynthesis of cholesterol (Merck index,10th Edition, entry no. 8619). Particularly preferred oil emulsions areoil in water emulsions, and in particular squalene in water emulsions.In addition, the most preferred oil emulsion adjuvants of the presentinvention comprise an antioxidant, which is preferably the oil.alpha.-tocopherol (vitamin E, EP 0 382 271 B1). WO 95/17210 and WO99/11241 disclose emulsion adjuvants based on squalene,alpha-tocopherol, and TWEEN® 80, optionally formulated with theimmunostimulants QS21 and/or 3D-MPL (which are discussed above). WO99/12565 discloses an improvement to these squalene emulsions with theaddition of a sterol into the oil phase. Additionally, a triglyceride,such as tricaprylin (C₂₇H₅₀O₆), may be added to the oil phase in orderto stabilize the emulsion (WO 98/56414).

The size of the oil droplets found within the stable oil in wateremulsion are preferably less than 1 micron, may be in the range ofsubstantially 30-600 nm, preferably substantially around 30-500 nm indiameter, and most preferably substantially 150-500 nm in diameter, andin particular about 150 nm in diameter as measured by photon correlationspectroscopy. In this regard, 80% of the oil droplets by number shouldbe within the preferred ranges, more preferably more than 90% and mostpreferably more than 95% of the oil droplets by number are within thedefined size ranges The amounts of the components present in the oilemulsions of the present invention are conventionally in the range offrom 2 to 10% oil, such as squalene; and when present, from 2 to 10%alpha tocopherol; and from 0.3 to 3% surfactant, such as polyoxyethylenesorbitan monooleate. Preferably the ratio of oil:alpha tocopherol isequal or less than 1 as this provides a more stable emulsion. Span 85may also be present at a level of about 1%. In some cases it may beadvantageous that the vaccines of the present invention will furthercontain a stabiliser.

The method of producing oil in water emulsions is well known to theperson skilled in the art. Commonly, the method comprises the mixing theoil phase with a surfactant such as a PBS/TWEEN80® solution, followed byhomogenization using a homogenizer. For instance, a method thatcomprises passing the mixture once, twice or more times through asyringe needle would be suitable for homogenizing small volumes ofliquid. Equally, the emulsification process in a microfluidiser (M110Smicrofluidics machine, maximum of 50 passes, for a period of 2 minutesat maximum pressure input of 6 bar (output pressure of about 850 bar))could be adapted to produce smaller or larger volumes of emulsion. Thisadaptation could be achieved by routine experimentation comprising themeasurement of the resultant emulsion until a preparation was achievedwith oil droplets of the required diameter.

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 GLA Aqueous Formulation

This example describes the preparation of a GLA-containing adjuvantaqueous formulation. The aqueous formulation of GLA (GLA-AF) containsWater For Injection (WFI), GLA (Avanti Polar Lipids, Inc., Alabaster,Ala.; product number 699800), and1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The formulationwas prepared by adding a solution of ethanol and POPC to a pre-weighedamount of GLA. This wetted GLA was sonicated for 10 minutes to dispersethe GLA as much as possible. The GLA was then dried under nitrogen gas.The dried GLA and POPC were reconstituted with WFI to the correctvolume. This solution was sonicated at 60° C. for 15-30 minutes untilall the GLA and POPC were in solution. For long term storage, GLA-AFformulations must be lyophilized. The lyophilization process consistedof adding glycerol to the solution until it was 2% of the total volume.Then the solution was placed in vials in 1-10 mL amounts. The vials werethen run through the lyophilization process which consisted of freezingthe solution and then putting it under vacuum to draw off the frozenwater by sublimation.

Example 2 GLA HPLC Analysis

This example describes HPLC analysis of a GLA-containing adjuvantaqueous formulation. After the formulation was manufactured (see Example1 above), certain release and stability tests were conducted to ensureproduct quality and reproducibility. All formulations were tested forrelease and long-term stability using High Performance LiquidChromatography (HPLC), Dynamic Light Scattering (DLS) and a visualexamination. HPLC chromatograms were collected using an Agilent 1100system and an ESA Corona CAD detector. The method was run using amethanol to chloroform gradient on a Waters Atlantis C18 column. Theinjections included 2.5 μg of GLA (Avanti Polar Lipids, Inc., Alabaster,Ala.; product number 699800, GLA-AF) or MPL® (GSK Biologicals,Rixensart, Belgium, MPL-AF) respectively, and 0.27 μg of syntheticphosphocholine (POPC) which was used as a solubilizing agent.

FIG. 1 shows HPLC data demonstrating the number and amounts ofcontaminating materials in MPL-AF and GLA-AF.

The HPLC profiles showed that GLA-AF was substantially purer thanMPL-AF. That is, there were fewer contaminant peaks in the GLA-AF thanin the MPL-AF adjuvant formulation. A purer starting product is oftremendous benefit to researchers as the biological response obtained isfrom the single major component used in the formulations of the GLA.

Example 3 GLA Oil Formulation

This example describes preparation of one milliliter of a GLA-containingadjuvant oil formulation. GLA (100 micrograms; Avanti Polar Lipids,Inc., Alabaster, Ala.; product number 699800) was emulsified in squalene(34.3 mg) with glycerol (22.7 mg), phosphotidylcholine or lecithin (7.64mg), Pluronic® F-68 (BASF Corp., Mount Olive, N.J.) or similar blockco-polymer (0.364 mg) in 25 millimolar ammonium phosphate buffer(pH=5.1) using 0.5 mg D,L-alpha-tocopherol as an antioxidant. Themixture was processed under high pressure until an emulsion formed thatdid not separate and that had an average particle size of less than 180nm. The emulsion was then sterile-filtered into glass unidose vials andcapped for longer term storage. This preparation may be used for atleast three years when stored at 2-8° C.

Example 4 GLA Stimulation of Murine Macrophages and Dendritic Cells

This example describes an in vitro model demonstrating an adjuvanteffect of GLA. Standard tissue culture methodologies and reagents wereemployed. Cells of the murine J774 and RAW267.4 macrophage cell line(American Type Culture Collection, Manassas, Va.) were maintainedaccording to the supplier's recommendations and cultured as adherentcell monolayers in multiwell dishes. Dendritic cells were derived frombone marrow progenitor cells following a protocol by Xiong et al. (J.Biol. Chem. 2004, 279, pp 10776-83). Various adjuvant concentrations ofsynthetic GLA (Avanti Polar Lipids, Inc., Alabaster, Ala.; productnumber 699800) were achieved by diluting an aqueous adjuvant preparationin cell culture medium (DMEM containing 10% fetal bovine serum), andcells were maintained for 24 hours at 37° C. in a humidified atmospherecontaining 5% CO₂, prior to collection of cell-free culturesupernatants. Supernatant fluids were assayed for soluble murinecytokines such as IL-12, IL-6, and TNF, and chemokines such as RANTES,using specific sandwich ELISA assay kits (eBiosciences, San Diego,Calif. for cytokines, and R&D Systems, Minneapolis, Minn. forchemokines) according to the manufacturer's instructions.

GLA-AF induced dose-dependent immune responses in mouse macrophage celllines and primary murine DC, characterized by the secretion of cytokinessuch as IL-12p40, IL-6, and TNF, and chemokines like RANTES.

Example 5 GLA Stimulation of Human Macrophages and Dendritic Cells

This example describes an in vitro model demonstrating the adjuvanteffects of GLA. Standard tissue culture methodologies and reagents wereemployed.

Cells of the human Mono Mac 6 macrophage cell line (American TypeCulture Collection, Manassas, Va.) were maintained according to thesupplier's recommendations and cultured as adherent cell monolayers inmultiwell plates. Dendritic cells were derived from peripheral bloodmononuclear cells (PBMC) following a standard protocol. Various adjuvantconcentrations of either synthetic GLA (Avanti Polar Lipids, Inc.,Alabaster, Ala.; product number 699800) or the natural product MPL® (GSKBiologicals, Rixensart, Belgium) were achieved by diluting an aqueousadjuvant preparation in cell culture medium (DMEM containing 10% fetalbovine serum, for MonoMac 6, or 10% human serum, for DC), and cells weremaintained for 24 hours at 37° C. in a humidified atmosphere containing5% CO₂, prior to collection of cell-free culture supernatants.Supernatant fluids were assayed for soluble human cytokines such asIL-1β, IL-23, and IL-6, and chemokines such as IP-10, RANTES and MIP-1 βusing specific sandwich ELISA assay kits (eBiosciences, San Diego,Calif. for cytokines, and Invitrogen, Carlsbad, Calif., for chemokines)according to the manufacturer's instructions.

FIG. 2 shows ELISA data demonstrating levels of cytokines and chemokinesexpressed by human macrophages of the Mono Mac 6 cell line (panels a-e),and monocyte-derived DC (panels f-h) in response to GLA stimulation.

GLA-AF induced a dose-dependent immune response in the human macrophagecell line Mono Mac 6 (FIG. 2, panels a-e), and primary DC (FIG. 2,panels f-h), characterized by the secretion of cytokines such as IL-1 β,IL-6, IL-23, and chemokines such as RANTES, IP-10, MIP-1β. GLA-AF wasactive at concentrations 5-500 lower compared to MPL-AF for all thecytokines and chemokines that were tested.

Example 6 GLA Stimulation of Human Blood Cells

This example describes an in vitro model demonstrating adjuvant effectsof GLA. Standard tissue culture methodologies and reagents wereemployed.

Human whole blood cells were cultured with various adjuvantconcentrations of either synthetic GLA (Avanti Polar Lipids, Inc.,Alabaster, Ala.; product number 699800) or the natural product MPL® (GSKBiologicals, Rixensart, Belgium), achieved by diluting an aqueousadjuvant preparation in cell culture medium (DMEM containing 10% fetalbovine serum). Blood cells were maintained for 16 hours at 37° C. in ahumidified atmosphere containing 5% CO₂, prior to collection ofcell-free culture supernatants. Supernatant fluids were assayed forsoluble human cytokine IL-1β using specific sandwich ELISA assay kit(eBiosciences, San Diego, Calif.) according to the manufacturer'sinstructions.

GLA-AF induced a dose-dependent immune response in human whole bloodcells, characterized by the secretion of IL-1β cytokine. In this assay,92 nM of GLA was equivalent in potency to 57,000 nM of MPL-AF.

Example 7 Use of GLA-Containing Vaccine In Vivo

This example describes an in vivo model demonstrating an adjuvant effectof GLA in a vaccine against Influenza. Standard immunologicalmethodologies and reagents were employed (Current Protocols inImmunology, Coligan et al. (Eds.) 2006 John Wiley & Sons, NY).

Mice (three Balb/c animals per group) were immunized twice at three-weekintervals with the Fluzone vaccine (Sanofi-Aventis, Swiftwater, Pa., at1/25 (20 μl) and 1/250 (2 μl) of the human dosage, alone, or formulatedin (i) an aqueous emulsion containing GLA (Avanti Polar Lipids, Inc.,Alabaster, Ala.; product number 699800; 20 μg per animal for eachimmunization) according to the procedure used in Example 1 above(“GLA-AF”), or (ii) a stable emulsion containing GLA (Avanti PolarLipids, Inc., Alabaster, Ala.; product number 699800; 20 μg per animalfor each immunization) according to the procedure used in Example 3above (“GLA-SE”). Sera were collected by bleeding animals one week aftereach immunization, and serum levels of total IgG antibodies specific forFluzone were examined by ELISA according to published methods (Id.).Serum levels of virus neutralizing antibodies were also examined byHemagglutination Inhibition Assay (HAI) according to published methods.

FIG. 3 shows ELISA data demonstrating levels of anti-Fluzone antibodyproduction induced in mice one week after each immunization (i.e., atday 7, panel A; and at day 28, panel B) using two different doses ofFluzone vaccine formulated with GLA-AF, or GLA-SE, compared to Fluzonealone. Means and SEM of reciprocal endpoint titers in each group/timepoint are shown. FIG. 3, panel C shows HAI data demonstrating levels ofvirus neutralizing antibody production induced in mice one week afterthe second immunization using two different doses of Fluzone vaccineformulated with GLA-AF, or GLA-SE, compared to Fluzone alone. Means andSEM of reciprocal endpoint titers in each group/time point are shown.

Total IgG and neutralizing antibody titers in response to Fluzonevaccination were enhanced by adding GLA, either in an aqueous or stableoil formulation. The adjuvanting effect of GLA was more pronounced withthe 2 μl dose of Fluzone vaccine, and induced antigen-specific humoralresponses similar to (GLA-AF) or greater than (GLA-SE) 20 μl of Fluzonevaccine alone. These results suggest that it is possible to reduce thedose of Fluzone vaccine by adjuvanting it with GLA-containingformulations, and still induce high levels of IgG and neutralizingantibody titers. This is of particular importance in the context of aworld pandemic infection such as Bird Flu.

Example 8 Use of GLA-Containing Vaccine In Vivo

This example describes an in vivo model demonstrating an adjuvant effectof GLA in a vaccine containing a specific Leishmania antigen. Standardimmunological methodologies and reagents were employed (CurrentProtocols in Immunology, Coligan et al. (Eds.) 2006 John Wiley & Sons,NY).

Mice (three C57BL/6 animals per group) were immunized three times atthree-week intervals with the SMT antigen (10 μg per animal for eachimmunization) used alone or formulated in a stable emulsion containingGLA (Avanti Polar Lipids, Inc., Alabaster, Ala.; product number 699800;20 μg per animal for each immunization) according to the procedure usedin Example 3 above, GLA-SE). Sera were collected by bleeding animals oneweek after the third immunization, and serum levels of IgG1 and IgG2cantibodies specific for SMT antigen were examined by ELISA according topublished methods.

FIG. 4 shows ELISA data demonstrating levels of anti-SMT antibodyproduction induced in mice one week after the third immunization usingSMT antigen alone, or formulated with GLA-SE. Means and SEM ofreciprocal endpoint titers in each group are shown.

Predominance of either IgG1 or IgG2c antibody isotype is associated withTH2 or TH1 responses respectively. It has been demonstrated that a TH1response is necessary for protection against Leishmania infection. SMTalone vaccination induced predominantly SMT-specific IgG1 antibody.SMT+GLA-SE vaccination induced higher antibody titers, and reverted thephenotype to a predominantly IgG2c antigen-specific antibody response,associated with protection against the disease.

Example 9 Use of GLA-Containing Vaccine In Vivo

This example describes an in vivo model demonstrating an adjuvant effectof GLA in a vaccine containing a specific Leishmania antigen. Standardimmunological methodologies and reagents were employed (CurrentProtocols in Immunology, Coligan et al. (Eds.) 2006 John Wiley & Sons,NY).

Mice (three Balb/c animals per group) were immunized three times attwo-week intervals with the Leish-110f antigen (10 μg per animal foreach immunization) formulated in a stable emulsion containing differentamounts of GLA (Avanti Polar Lipids, Inc., Alabaster, Ala.; productnumber 699800; 40, 20, 5, or 1 μg per animal for each immunizationaccording to the procedure used in Example 3 above, GLA-SE). Sera werecollected by bleeding animals one week after the first immunization, andserum levels of IgG1 and IgG2a antibodies specific for Leish-110f wereexamined by ELISA according to published methods (Id.).

FIG. 5 shows ELISA data demonstrating levels of anti-Leish-110f antibodyproduction induced in mice one week after the first immunization usingLeish-110f antigen formulated with different amounts of GLA (40, 20, 5,or 1 μg), compared to saline controls. Means and SEM of reciprocalendpoint titers in each group are shown.

Leish-110f-specific IgG1 and IgG2a antibody titers were GLAdose-dependent. Predominance of TH1 associated IgG2a antibody wasobserved at all concentrations of GLA tested.

Example 10 Use of GLA-Containing Vaccine In Vivo

This example describes an in vivo model demonstrating an adjuvant effectof GLA in a vaccine containing a specific Leishmania antigen. Standardimmunological methodologies and reagents were employed (CurrentProtocols in Immunology, Coligan et al. (Eds.) 2006 John Wiley & Sons,NY).

Mice (three Balb/c animals per group) were immunized three times atthree-week intervals with saline or the Leish-111f antigen (10 μg peranimal for each immunization) formulated in a stable emulsion containingGLA (Avanti Polar Lipids, Inc., Alabaster, Ala.; product number 699800;20 μg per animal for each immunization, according to the procedure usedin Example 3 above, GLA-SE). Two weeks after the last injection, micewere sacrificed and spleen collected to analyze T cell-dependent IFN-γand IL-4 cytokine responses to in vitro antigen stimulation by ELISAaccording to published methods.

Predominance of either IL-4 or IFN-γ cytokine is associated with TH2 orTH1 responses respectively. We and others have demonstrated that a TH1response is necessary for protection against Leishmania infection. Allanimals responded well to ConA, a potent mitogen. Leish-111f+GLA-SEvaccination induced Leish-111f antigen-specific cytokine responses whileno such responses were observed in the saline control group. Whencompared to ConA, Leish-111f+GLA-SE vaccination induced much more IFN-γthan IL-4, a TH1:TH2 ratio or phenotype associated with protectionagainst the disease.

Example 11 Use of GLA-Containing Vaccine In Vivo

This example describes an in vivo model demonstrating an adjuvant effectof GLA in a vaccine containing a specific Leishmania antigen. Standardimmunological methodologies and reagents were employed (CurrentProtocols in Immunology, Coligan et al. (Eds.) 2006 John Wiley & Sons,NY).

Mice (three Balb/c animals per group) were immunized three times attwo-week intervals with saline or the Leish-110f antigen (10 μg peranimal for each immunization) formulated in a stable emulsion containingdifferent amounts of (i) GLA (Avanti Polar Lipids, Inc., Alabaster,Ala.; product number 699800; 40, 5, or 1 μg per animal for eachimmunization) according to the procedure used in Example 3 above(GLA-SE), or (ii) MPL® (40, 5, or 1 μg per animal for each immunization)in an emulsion as supplied by the manufacturer (“MPL-SE”, GSKBiologicals, Rixensart, Belgium). One week after the last injection,mice were sacrificed and spleen collected to analyze T cell-dependentIFN-γ cytokine responses to in vitro antigen stimulation by ELISAaccording to published methods (Id.). IFN-γ cytokine responses have beenassociated with a TH1 protective phenotype against Leishmania infection.

FIG. 6 shows ELISA data demonstrating levels of anti-Leish-110f IFN-γcytokine production induced in mice one week after the thirdimmunization using Leish-110f antigen formulated with different amountsof GLA, compared to saline controls. Means and SEM in each group areshown.

All animals responded well to ConA, a potent cell activator and mitogen.Leish-110f+GLA-SE vaccination induced Leish-110f antigen-specificcytokine responses, in a dose-dependent manner, while no such responseswere observed in the saline control group. At all concentration tested,GLA-SE was more potent than MPL-SE, in inducing higher levels of IFN-γsecreted by antigen-specific T cells

In conclusion, the addition of GLA in a stable oil formulation toLeishmania vaccine antigen candidate Leish-110f induced predominantlyantigen-specific immune responses of the cellular type (T cell)associated with the protective TH1 phenotype. In addition, GLA-SE wasmore potent than MPL-SE in inducing protection-associated cytokines likeIFN-γ.

Example 12 Use of GLA-Containing Vaccine In Vivo

This example describes an in vivo model demonstrating an adjuvant effectof GLA in a vaccine containing a specific Leishmania antigen. Standardimmunological methodologies and reagents were employed (CurrentProtocols in Immunology, Coligan et al. (Eds.) 2006 John Wiley & Sons,NY).

Mice (three Balb/c animals per group) were immunized three times attwo-week intervals with saline or the Leish-110f antigen (10 μg peranimal for each immunization) formulated in a stable emulsion containingdifferent amounts of (i) GLA (Avanti Polar Lipids, Inc., Alabaster,Ala.; product number 699800; 20 μg or 5 μg per animal for eachimmunization) according to the procedure used in Example 3 above(GLA-SE), or (ii) MPL® (20 μg or 5 μg per animal for each immunization)in an emulsion as supplied by the manufacturer (“MPL-SE”, GSKBiologicals, Rixensart, Belgium). One week after the last injection,mice were sacrificed and spleen collected to analyze T cell-dependentIFN-γ, IL-2, and TNF cytokine responses to in vitro antigen stimulationby intracellular cell staining (ICS) and Flow cytometry according topublished methods (Id.). These three cytokines have been associated witha TH1 protective phenotype against Leishmania infection.

When analyzed at the single cell level, the frequency of CD4+ T cellsexpressing all three cytokines IFN-γ, IL-2, and TNF or a combination ofIFN-γ and IL-2 was higher in the Leish-110f+GLA-SE group compared to theLeish-110f+MPL-SE group, and this was observed at both 20 and 5 μgdoses. It has been reported (Seder et al.) that high frequencies of CD4+T cells expressing all three cytokines IFN-γ, IL-2, and TNF correlateswith protection against Leishmania infection.

In conclusion, the addition of GLA in a stable oil formulation toLeishmania vaccine antigen candidate Leish-110f induced predominantlyantigen-specific immune responses of the cellular type (T cell)associated with the protective TH1 phenotype. In addition, GLA-SE wasmore potent than MPL-SE in inducing protection-associated cytokines likeIFN-γ, IL-2, and TNF.

Example 13 Use of GLA-Containing Vaccine In Vivo

This example describes an in vivo model demonstrating an adjuvant effectof GLA in a vaccine containing a specific Mycobacterium tuberculosisantigen. Standard immunological methodologies and reagents were employed(Current Protocols in Immunology, Coligan et al. (Eds.) 2006 John Wiley& Sons, NY).

Mice (three C57BL/6 animals per group) were immunized three times atthree-week intervals with the ID83 antigen (8 μg per animal for eachimmunization) used alone or formulated in a stable emulsion containingGLA (Avanti Polar Lipids, Inc., Alabaster, Ala.; product number 699800;20 μg per animal for each immunization, according to the procedure usedin Example 3 above, GLA-SE). Sera were collected by bleeding animals oneweek after the third immunization, and serum levels of IgG1 and IgG2cantibodies specific for ID83 were examined by ELISA according topublished methods (Id.) Predominance of either IgG1 or IgG2c antibodyisotype is associated with TH2 or TH1 responses, respectively. It hasbeen demonstrated that a TH1 response is necessary for protectionagainst Mycobacterium tuberculosis infection.

Vaccination with ID83 alone induced predominantly antigen-specific IgG1antibody. In contrast, ID83+ GLA-SE vaccination induced higher antibodytiters, and reverted the phenotype to a predominantly IgG2cantigen-specific antibody response, associated with protection againstthe disease.

Example 14 Use of GLA-Containing Vaccine In Vivo

This example describes an in vivo model demonstrating an adjuvant effectof GLA in a vaccine containing a specific Mycobacterium tuberculosisantigen. Standard immunological methodologies and reagents were employed(Current Protocols in Immunology, Coligan et al. (Eds.) 2006 John Wiley& Sons, NY).

Mice (three C57BL/6 animals per group) were immunized three times atthree-week intervals with the ID83 antigen (8 μg per animal for eachimmunization) used alone or formulated in a stable emulsion containingGLA (GLA-SE), GLA+CpG (CpG₁₈₂₆, Coley Pharmaceuticals, 25 μg)(GLA/CpG-SE), or GLA+Gardiquimod (GDQ) (Invivogen, 20 μg) (GLA/GDQ-SE).Three weeks after the last injection, mice were sacrificed and spleenscollected to analyze CD4+ and CD8+ T cell-dependent IFN-γ, IL-2, and TNFcytokine responses to in vitro ID83 antigen stimulation by ICS and Flowcytometry according to published methods. Expression of IFN-γ, IL-2, andTNF cytokines have been associated with protective TH1 responses againstM. tuberculosis infection.

FIG. 7 shows ICS data demonstrating the frequencies of ID83-specificIFN-γ, IL-2, and TNF cytokine producing CD4+ and CD8+ T cells induced inmice one week after the third immunization using ID83 alone oradjuvanted with formulations containing GLA (GLA-SE), GLA+CpG(GLA/CpG-SE), or GLA+GDQ (GLA/GDQ-SE).

Frequencies of ID83 specific cytokine producing CD4+ or CD8+ T cellswere at background levels for the saline and ID83 alone vaccine groups.ID83 antigen specific cytokine producing T cells, both CD4+ and CD8+,were induced by ID83+GLA-SE vaccination, and their frequency furtherincreased by the addition of a second TLR ligand like GDQ (TLR7/8) orCpG (TLR9). T cells expressing IFN-γ+TNF or IFN-γ+IL-2 were thepredominant populations.

In conclusion, adjuvanting an antigen against M. tuberculosis withGLA-SE greatly enhanced the antigen specific cellular response (T cells)as measured by the frequencies of T cells expressing IFN-γ, IL-2, and/orTNF cytokines. Combining GLA-SE with another TLR ligand furtherincreased the frequency of antigen specific cytokine producing cells, aphenotype associated with protection against this disease.

Example 15 Use of GLA-Containing Vaccine In Vivo

This example describes an in vivo model demonstrating an adjuvant effectof GLA in a vaccine containing a specific Mycobacterium leprae antigen.Standard immunological methodologies and reagents were employed (CurrentProtocols in Immunology, Coligan et al. (Eds.) 2006 John Wiley & Sons,NY).

Mice (three C57BL/6 animals per group) were immunized three times atthree-week intervals with the ML0276 antigen (10 μg per animal for eachimmunization) adjuvanted with aqueous formulations containing CpG(CpG₁₈₂₆, Coley Pharmaceutical, 25 μg per animal for each immunization),or Imiquimod (IMQ) (3M Pharma, 25 μg per animal for each immunization),or GLA (Avanti Polar Lipids, Inc., Alabaster, Ala.; product number699800; 25 μg per animal for each immunization according to theprocedure used in Example 3 above, GLA-SE), a mix of the three, orsaline as negative control. Sera were collected by bleeding animalsthree weeks after the second immunization, and serum levels of IgGantibodies specific for ML0276 were examined by ELISA according topublished methods (Id.).

Animals from the saline control group did not show ML0276 specific IgG,and those from the ML0276+CpG and ML0276+IMQ groups showed a very lowlevel of antigen specific antibody. In contrast, ML0276+GLA-SE induced asignificant level of ML0276 specific IgG, that was further increasedwhen the three adjuvants were used together.

In conclusion, the data support the adjuvanting effect of GLA-SE and/ora combination of GLA-SE with additional TLR ligands when used withantigen ML0276 for the induction of antigen specific antibodies.

Example 16 Use of GLA-Containing Vaccine In Vivo

This example describes an in vivo model demonstrating an adjuvant effectof GLA in a vaccine containing a specific Mycobacterium leprae antigen.Standard immunological methodologies and reagents were employed (CurrentProtocols in Immunology, Coligan et al. (Eds.) 2006 John Wiley & Sons,NY).

Mice (three C57BL/6 animals per group) were immunized three times atthree-week intervals with the ML0276 antigen (10 μg per animal for eachimmunization) adjuvanted with aqueous formulations containing CpG(CpG₁₈₂₆, Coley Pharmaceutical, 25 μg per animal for each immunization),or Imiquimod (IMQ) (3M Pharma, 25 μg per animal for each immunization),or GLA (Avanti Polar Lipids, Inc., Alabaster, Ala.; product number699800; 25 μg per animal for each immunization, according to theprocedure used in Example 3 above, GLA-SE), a mix of the three, orsaline as negative control. Three weeks after the last injection, micewere sacrificed and spleen collected to analyze CD4+T cell-dependentIFN-γ cytokine responses to in vitro ML0276 antigen stimulation by ICSand Flow cytometry according to published methods. Expression of IFN-γcytokine has been associated with protective TH1 responses against M.leprae infection.

FIG. 8, panel A shows ICS data demonstrating the frequencies ofML0276-specific IFN-γ cytokine producing CD4+ T cells induced in miceone week after the third immunization using ML0276 antigen formulatedwith aqueous formulations containing CpG, or Imiquimod (IMQ), or astable oil emulsion containing GLA (GLA-SE), or the three mixedtogether, compared to saline and naïve controls. Means in each group areshown. FIG. 8, panel B shows data demonstrating the cellularity of lymphnodes draining the site of M. leprae infection in mice immunized withML0276 antigen formulated with aqueous formulations containing CpG, orImiquimod (IMQ), or a stable oil emulsion containing GLA (GLA-SE), or amixture of the three, compared to saline and naïve controls. Means andSEM in each group are shown.

Animals from the saline control group did not show ML0276 specific IFN-γresponses with a background frequency of 0.04% positive cells. Thosefrom the CpG and IMQ groups showed a slightly increased frequency ofantigen specific cytokine producing cells with 0.17% and 0.11%respectively. In contrast, a significantly higher number of ML0276specific IFN-γ+ CD4+ T cells (0.66%) were observed when GLA-SE was usedas an adjuvant, a frequency that was further increased when the threeadjuvants were mixed together (2.14%).

A subset of mice was subsequently challenged with M. leprae and found tobe protected by ML0276+GLA-SE as measured by the reduction in the numberof cells in the lymph nodes draining the site of challenge as comparedto infected saline controls. Vaccination with ML0276+CpG and ML0276+IMQinduced only a modest decrease in cell numbers compared to saline.

In conclusion, the data support the adjuvanting effect of GLA-SE and/ora combination of GLA-SE with additional TLR ligands when used withantigen ML0276 for the induction of antigen specific cellular responses.

Example 17 GLA Stimulation of Human Dendritic Cells

This example describes an in vitro model demonstrating an adjuvanteffect of GLA. Standard tissue culture methodologies and reagents wereemployed.

Dendritic cells were derived from purified blood CD14+ monocytesfollowing a published protocol. Various adjuvant concentrations ofeither synthetic GLA (Avanti Polar Lipids, Inc., Alabaster, Ala.;product number 699800) or the natural product MPL® (GSK Biologicals,Rixensart, Belgium) were achieved by diluting an aqueous adjuvantpreparation in cell culture medium (RPMI containing 5% human serum).Prior to collection and analysis of the expression of activationmarkers, cells were maintained for 44 hours at 37° C. in a humidifiedatmosphere containing 5% CO₂. Expression levels of the costimulatorymolecule CD86 at the surface of DC were used as an indicator of cellactivation and measured by flow cytometry on a LSRII instrument (BDBiosciences, San Jose, Calif.) using CD86-specific fluorochrome-labeledantibody (eBiosciences, San Diego, Calif.) according to themanufacturer's instructions.

FIG. 9 shows data obtained by flow cytometry demonstrating levels ofCD86 molecule expressed at the surface of human DC in response to 44 hof stimulation with 10,000 ng/ml to 0.010 ng/ml GLA (panel A) or MPL(panel B). A positive stimulation control consisting of PGE2, IL-1β,TNF, and IL-6 was also included.

GLA-AF induced a dose-dependent immune response in the human primary DC(FIG. 9, panel A), characterized by the increased expression of CD86.Maximal CD86 expression was seen with GLA at 10,000 ng/ml, 1000 ng/ml,and 100 ng/ml, while MPL had only maximal expression at 10,000 ng/ml and1000 ng/mL with DCs generated from Donor N003. An additional threedonors were used to generate DC cultures and evaluated in a similarmanner with GLA and MPL stimulation at 1000 ng/ml to 1 ng/mL, see FIG.10. The dendritic cells from these donors showed different levels ofsensitivity, but in every case lower doses of GLA achieved higher CD86expression than MPL. This demonstrated that GLA was active atconcentrations at least 10-fold lower compared to MPL for humandendritic cell maturation.

Example 18 Use of GLA-Containing Vaccine In Vivo

This example describes an in vivo model demonstrating an adjuvant effectof GLA in a vaccine containing a specific Leishmania antigen. Standardimmunological methodologies and reagents were employed (CurrentProtocols in Immunology, Coligan et al. (Eds.) 2006 John Wiley & Sons,NY).

Four Balb/c mice per treatment group were immunized three times attwo-week intervals either with saline or the Leish-110f antigen (10 μgper animal for each immunization) formulated in a stable emulsioncontaining 20 μg of (i) GLA (Avanti Polar Lipids, Inc., Alabaster, Ala.;product number 699800; per animal for each immunization) according tothe procedure used in Example 3 above (GLA-SE), or (ii) MPL® in anemulsion as supplied by the manufacturer (“MPL-SE”, GSK Biologicals,Rixensart, Belgium, per animal for each immunization). Three weeks afterthe last injection, mice were challenged intradermally in the pinea ofboth ears with 2×10³ purified Leishmania major clone V1(MOHM/IL/80/Friedlin) metacyclic promastigotes according to publishedmethods (Id.). Development of cutaneous lesions was monitored weekly for6 weeks post-infection.

FIG. 11 shows diameter of lesions in mice immunized using Leish-110fantigen formulated with GLA-SE or MPL-SE, compared to saline controls.Means in each group are shown.

All animals in the saline control groups developed progressivenon-healing lesions. Ear lesions that started to develop during thefirst 2 weeks, were controlled at week 3, and eventually resolved byweek 4 in mice immunized with Leish-110f+GLA-SE. Lesions of miceimmunized with Leish-110f+MPL-SE were reduced compared to the salinegroup but did not resolve. GLA-SE was more potent than MPL-SE inpreventing the development of lesions.

In conclusion, the addition of GLA in a stable oil formulation toLeishmania vaccine antigen candidate Leish-110f prevented thedevelopment of cutaneous lesions upon challenge with Leishmania majorparasites. In addition, GLA-SE was more potent than MPL-SE incontrolling lesion development.

Example 19 Use of GLA-Containing Vaccine In Vivo

This example describes an in vivo model demonstrating an adjuvant effectof GLA in a vaccine containing a specific Leishmania antigen. Standardimmunological methodologies and reagents were employed (CurrentProtocols in Immunology, Coligan et al. (Eds.) 2006 John Wiley & Sons,NY).

Four Balb/c mice per treatment group were immunized three times attwo-week intervals with saline or the Leish-110f antigen (10 μg peranimal for each immunization) formulated in a stable emulsion containing20 μg of (i) GLA (Avanti Polar Lipids, Inc., Alabaster, Ala.; productnumber 699800; per animal for each immunization) according to theprocedure used in Example 3 above (GLA-SE), or (ii) MPL® in an emulsionas supplied by the manufacturer (“MPL-SE”, GSK Biologicals, Rixensart,Belgium, per animal for each immunization). Three weeks after the lastinjection, mice were challenged intradermally in the pinea of both earswith 2×10³ purified Leishmania major clone V1 (MOHM/IL/80/Friedlin)metacyclic promastigotes according to published methods (Id.). Parasiteburden in the ear and draining lymph nodes of infected mice weredetermined 6 weeks post-infection.

FIG. 12 shows the number of parasites recovered in the ear and draininglymph nodes of mice immunized using Leish-110f antigen formulated withGLA-SE or MPL-SE, compared to saline controls. Number of parasites inindividual organs and means in each group are shown. Differences inparasite numbers between groups were evaluated for statisticalsignificance using the Student's t test. A difference was consideredsignificant when the p value was <0.05.

Animals in the saline control groups showed an average parasite load of10⁵ and 5×10⁴ in ears and draining lymph nodes respectively. In miceimmunized with Leish-110f+GLA-SE, no parasites could be detected in 8/8ears and in 6/8 draining lymph nodes. In contrast, in mice immunizedwith Leish-110f+MPL-SE, no parasites could be detected in 4/8 ears and2/8 draining lymph nodes. GLA-SE was more potent than MPL-SE incontrolling infection with Leishmania major.

In conclusion, the addition of GLA in a stable oil formulation toLeishmania vaccine antigen candidate Leish-110f controlled parasiteburden in both ear and draining lymph nodes upon challenge withLeishmania major parasites. In addition, GLA-SE was more potent thanMPL-SE in controlling the number of parasites recovered from theinfected tissues.

Example 20 Use of GLA-Containing Vaccine In Vivo

This example describes an in vivo model demonstrating an adjuvant effectof GLA in a lentiviral vaccine containing OVA antigen. Standardimmunological methodologies and reagents were employed (CurrentProtocols in Immunology, Coligan et al. (Eds.) 2006 John Wiley & Sons,NY).

Five C57BL/6 mice per treatment group were immunized one time withsaline, the lentiviral vaccine or the lentiviral vaccine administered atthe same time as a stable emulsion containing 20 μg of GLA (Avanti PolarLipids, Inc., Alabaster, Ala.; product number 699800; per animal foreach immunization) according to the procedure used in Example 3 above(GLA-SE). Two weeks after the injection, splenocytes were removed and Tcell immunogenicity (phenotype and effector functions) was evaluated bymultiparameter flow cytometry.

Splenocytes were plated in 24-well plates and stimulated ex vivoovernight with 2 μg/ml anti-CD28 (eBioscience, San Diego, Calif.),anti-CD49d (eBioscience), and 20 μg/ml OVA as antigen (or withPMA/ionomycin, or without antigen as a negative control) in completemedia at 37° C. After a 2 hour incubation at 37° C., brefeldin A(GolgiPlug: BD Biosciences, San Jose, Calif.) was added to the wells andthe incubation resumed for an additional 12 hrs at 37° C. Cells wereblocked with anti-CD16/32 (eBioscience) 1:50 in 50 μl and then stainedwith AlexaFluor 700-anti-CD3 (eBioscience), PerCP-anti-CD4 (BDBiosciences), and PE-anti-CD8 (BD Biosciences). The cells were fixedusing the Cytofix/Cytoperm kit (BD Biosciences) and intracellularstaining was performed according to the BD Biosciences protocol. Cellswere blocked with anti-CD16/32 and intracellularly stained withFITC-anti-TNF-α (eBioscience), Pacific Blue-anti-IL-2 (eBioscience) andPE-Cy7-anti-IFN-γ (BD Biosciences). Cells were analyzed with a LSRIIFACS machine (BD Biosciences) and the DIVA software to quantify CD4+Tcells producing IFN-γ, IL-2 and TNF-α.

FIG. 13 shows CD8 (panels A-C) and CD4 (panels D-F) responses in miceimmunized with the lentivirus vaccine expressing OVA antigen in thepresence and absence of GLA-SE, compared to saline controls. Means ineach group are shown.

Splenocytes from animals in the lentivector vaccine plus GLA-SE hadhigher percentages of CD8+CD44+ as well as CD4+CD44+ cytokine secretingcells than splenocytes from animals receiving the lentivaccine alone.The addition of GLA-SE to the lentivaccine immunization resulted in anincreased frequency of antigen-specific triple cytokine (IFN-γ, TNF-α,IL-2) producing T cells.

In conclusion, the addition of GLA in a stable oil formulation to thelentiviral vector vaccine improved both the quantity and quality ofeffector T cells which may have an essential role in protection againsta variety of infections.

Example 21 Use of GLA-Containing Vaccine In Vivo

This example describes an in vivo model demonstrating an adjuvant effectof GLA-SE in a vaccine against Influenza. Standard immunologicalmethodologies and reagents were employed (e.g., Current Protocols inImmunology, Coligan et al. (Eds.) 2006 John Wiley & Sons, NY).

Three Balb/c mice treatment group were immunized i.d. with microneedles(NanoPass Technologies Ltd., Nes Ziona, Israel) 2 times at 3 weekintervals (total volume was 100 μl; 50 μl per hind leg) with i) a stableemulsion containing GLA, “GLA-SE”, (Avanti Polar Lipids, Inc.,Alabaster, Ala.; product number 699800; 5 μg per animal for eachimmunization) and Fluzone® (Sanofi Pasteur, Stillwater Pa.; 2 μg and 0.2μg treatment groups); ii) stable emulsion (SE) plus Fluzone; or iii)Fluzone only. Mice were immunized with the 2006-2007 commercial Fluzoneformula which includes components from A/New Calcdonia/20/99 (H1N1),A/Wisconsin/67/2005 (H3N2), and B/Malaysia/2506/2004. Mice were bled oneweek after the second immunization and serum was analyzed for antibodyresponses to Fluzone.

IgG2a responses were dominant in mice immunized using Fluzone combinedwith GLA-SE. One week following a boost immunization, significantlygreater levels of anti-Fluzone antibodies (total IgG and IgG2a) wereinduced in this treatment group (see FIG. 14, panels A-B). IgG1 antibodylevels were similar in all treatment groups (see FIG. 14, panel C). Asignificant increase in endpoint antibody titers were seen in mice thatreceived a boost immunization of Fluzone plus GLA-SE as compared to micethat received a boost immunization of Fluzone vaccine alone (e.g., FIG.14, compare “GLA-SE” treatment group (Fluzone plus GLA-SE) with “(−)”treatment group (Fluzone)). No differences in antibody titers wereobserved in mice that received a boost immunization of Fluzone combinedwith a stable emulsion (SE) as compared to mice that received a boostimmunization of Fluzone vaccine alone (e.g., FIG. 14, compare “SE”treatment group (Fluzone plus SE) with “(−)” treatment group (Fluzone)).

Total IgG and IgG2a antibody titers were enhanced in response to Fluzonevaccination combined with GLA-SE. The adjuvanting effect of GLA-SE wassimilar for both the 2 μg and 0.2 μg doses of Fluzone vaccine incombination with GLA-SE. These results suggest that it is possible toreduce the dose of Fluzone vaccine by adjuvanting it with GLA-SE, andstill induce high levels of IgG and IgG2a antibody titers.

Example 22 Use of GLA-Containing Vaccine In Vivo

This example describes an in vivo model demonstrating an adjuvant effectof GLA-SE in a vaccine against Influenza. Standard immunologicalmethodologies and reagents were employed (Current Protocols inImmunology, Coligan et al. (Eds.) 2006 John Wiley & Sons, NY).

Three Balb/c mice treatment group were immunized i.m. 2 times at 3 weekintervals (total volume was 100 μl; 50 μl per hind leg) with i) a stableemulsion containing GLA, “GLA-SE”, (Avanti Polar Lipids, Inc.,Alabaster, Ala.; product number 699800; 5 μg per animal for eachimmunization) and Fluzone® (Sanofi Pasteur, Stillwater Pa.; 0.2 μg); ii)stable emulsion (SE) plus Fluzone; iii) Fluzone only; or iv) saline.Mice were immunized with the 2006-2007 commercial Fluzone formula whichincludes components from A/New Calcdonia/20/99 (H1N1),A/Wisconsin/67/2005 (H3N2), and B/Malaysia/2506/2004. Spleens wereharvested three weeks following an i.m. boost with saline, Fluzonealone, Fluzone plus SE, or Fluzone plus GLA-SE.

Splenocytes were plated in 48-well plates and were stimulated ex vivowith H1N1 antigen (Influenza virus A H1N1, New Calcdonia, FitzgeraldIndustries, Concord Mass.) or media (e.g., negative control) andsupernatants were collected 72 hours later. Supernatants were assayedfor soluble murine cytokines including IFN-gamma, IL-2, IL-10 and IL-5,using specific sandwich ELISA assay kits (eBiosciences, San Diego,Calif.) according to the manufacturer's instructions.

Mice immunized using Fluzone (0.2 μg) plus GLA-SE (5 μg) hadsignificantly higher levels of IFN-γ and IL-2 cytokines following H1N1antigen stimulation of splenocytes as compared to unstimulated controls(FIG. 15). Furthermore, treatment groups ii-iv failed to showsignificant increases in IFN-γ and IL-2 after H1N1 antigen stimulation.In contrast, mice immunized with Fluzone (0.2 μg) and SE displayed adifferent cytokine profile e.g., significantly higher levels of IL-5 andIL-10 as compared to other treatment groups.

These results indicate that mice immunized with Fluzone plus GLA-SEinduced a significant Th₁ type T-cell cytokine response compared toother treatment groups. Th₁ cytokine responses are believed have anessential role in protection against a variety of infections.

Example 23 USP Rabbit Pyrogen Test

GLA and MPL were evaluated for pyrogenicity/toxicity using the UnitedStates Pharmacopeial (USP) rabbit pyrogen test method. GLA was tested atthe same doses and conditions commonly used for MPL. Both GLA and MPLpassed the USP<151> requirements for the absence of pyrogens at thedoses evaluated. These data indicate that the unexpected high potency ofGLA compared to MPL is not associated with increased toxicity.Accordingly, lower doses of GLA can be used to achieve the same orimproved potency relative to MPL while also having correspondinglyreduced pyrogenicity due to the lower doses needed.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1-34. (canceled)
 35. A pharmaceutical composition for inducing or enhancing an immune response against herpes simplex virus comprising: (a) at least one herpes simplex virus antigen; and (b) a lipid adjuvant of the formula:

wherein: R¹, R³, R⁵ and R⁶ are C₁₁-C₂₀ alkyl; and R² and R⁴ are C₁₂-C₂₀ alkyl; or a pharmaceutically acceptable salt thereof.
 36. The composition of claim 35, wherein the at least one herpes simplex virus antigen comprises at least one HSV-1 or HSV-2 antigen.
 37. The composition of claim 35, wherein the at least one herpes simplex virus antigen comprises whole live or inactivated virus.
 38. The composition of claim 35, wherein the at least one herpes simplex virus antigen comprises at least one purified or recombinant herpes simplex virus antigen selected from the group consisting of an HSV immediate early protein, an HSV ICP27 protein and an HSV gD protein, or combinations thereof.
 39. The composition of claim 35, wherein the alkyl is straight chain saturated alkyl.
 40. The composition of claim 35, wherein R¹, R³, R⁵ and R⁶ are undecyl and R² and R⁴ are tridecyl.
 41. The composition of claim 35, wherein the lipid adjuvant is synthetic.
 42. The composition of claim 35, wherein the lipid adjuvant is at least 80% pure based on the total weight of lipid adjuvant species in the composition.
 43. The composition of claim 35, wherein the lipid adjuvant is at least 95% pure based on the total weight of lipid adjuvant species in the composition.
 44. The composition of claim 35, further comprising a pharmaceutically acceptable carrier or excipient.
 45. The composition of claim 35, which is in a form of an oil-in-water emulsion, a water-in-oil emulsion, a liposome, or a microparticle.
 46. The composition of claim 35, which is in a form of an oil-in-water emulsion and the oil is a metabolizable oil.
 47. The composition of claim 46, wherein the oil is squalene.
 48. The composition of claim 35, further comprising an antioxidant.
 49. The composition of claim 35, further comprising alpha-tocopherol.
 50. The composition of claim 35, further comprising oil and alpha-tocopherol where the oil:alpha-tocopherol are present in a ratio of equal to or less than
 1. 51. The composition of claim 35, which has been sterile filtered.
 52. The composition of claim 35, which is in the form of a stable aqueous suspension of less than 0.2 um and further comprises at least one component selected from the group consisting of phospholipids, fatty acids, surfactants, detergents, saponins, and fluorodated lipids.
 53. A method for inducing or enhancing an immune response against a herpes simplex virus comprising the steps of administering to a subject in need thereof a composition of any one of claims 35-52. 